Protein
Proteins are complex substances found in all living
organisms. They are very important for nutrition and help carry out many
chemical processes that keep the body alive.
The importance of proteins was discovered in the early 19th
century. In 1838, a Swedish chemist named Jöns Jacob Berzelius gave them the
name “protein,” which comes from a Greek word meaning “most important.”
Proteins are different in every species, meaning each type
of living organism has its own unique proteins. Even within the same organism,
proteins vary in different parts of the body. For example, proteins in muscles
are different from those in the brain and liver.
A protein molecule is very large compared to sugar or salt.
It is made up of many small units called amino acids joined together in
long chains, like beads on a string. There are about 20 different amino
acids found in proteins. The order and type of these amino acids decide how
a protein looks and works.
Proteins with similar functions usually have similar
structures. Although scientists cannot fully explain every function of a
protein yet, they know that its structure is closely related to its function.
Plants can make all the amino acids they need using simple
substances like carbon dioxide and minerals through photosynthesis. However,
animals (including humans) cannot make all amino acids, so they must get them
from food such as meat, milk, eggs, and some plant sources like legumes.
Different parts of the body contain different amounts of
protein. For example, muscles have about 30% protein, and the liver has around
20–30%. However, the importance of proteins is not based on quantity but on
their function.
Proteins perform many important roles in the body:
- Enzymes:
Help speed up chemical reactions necessary for life
- Hormones:
Control and regulate body activities
- Hemoglobin:
Carries oxygen in the blood
- Structural
proteins: Give shape and support to the body
Without proteins, especially enzymes, life would not be
possible.
General structure and properties of proteins
The amino acid composition of proteins
The common property of all proteins is that they consist of
long chains of α-amino (alpha amino) acids. The α-amino acids are so
called because the α-carbon atom in the molecule carries an
amino group (―NH2); the α-carbon atom also carries a carboxyl group
(―COOH).
In acidic solutions, when the pH is less than 4, the ―COO
groups combine with
hydrogen ions (H+) and are thus converted into the uncharged form
(―COOH). In alkaline solutions, at pH above 9, the ammonium groups (―NH+3)
lose a hydrogen
ion and are converted into amino groups (―NH2). In the pH
range between 4 and 8, amino acids carry both a positive and a negative charge
and therefore do not migrate in an electrical field. Such structures have been
designated as dipolar ions, or zwitterions
(i.e., hybrid ions).
Although more than 100 amino acids occur in nature,
particularly in plants, only 20 types are commonly found in most proteins. In
protein molecules the α-amino acids are linked to each other by peptide bonds between
the amino group of one amino acid and
the carboxyl group of its neighbor.
The condensation (joining)
of three amino acids yields the tripeptide.
It is customary to write the structure of peptides in
such a way that the free α-amino group (also called the N terminus of the
peptide) is at the left side and the free carboxyl group (the C terminus) at
the right side. Proteins are macromolecular polypeptides—i.e.,
very large molecules (macromolecules)
composed of many peptide-bonded amino acids. Most of the common ones contain
more than 100 amino acids linked to each other in a long peptide chain. The
average molecular
weight (based on the weight of a hydrogen atom as 1)
of each amino acid is approximately 100 to 125; thus, the molecular weights of
proteins are usually in the range of 10,000 to 100,000 daltons (one dalton is
the weight of one hydrogen atom). The species-specificity and organ-specificity
of proteins result from differences in the number and sequences of amino acids.
Twenty different amino acids in a chain 100 amino acids long can be arranged in
far more than 10100 ways (10100 is the number
one followed by 100 zeroes).
Structures of common amino acids
The amino acids present in proteins differ from each other
in the structure of their side (R) chains. The simplest amino acid
is glycine,
in which R is a hydrogen atom. In a number of amino
acids, R represents straight or branched carbon chains.
One of these amino acids is alanine, in which R is
the methyl group (―CH3). Valine, leucine, and isoleucine, with
longer R groups, complete the alkyl side-chain series. The
alkyl side chains (R groups) of these amino acids are nonpolar;
this means that they have no affinity for water but some
affinity for each other. Although plants can form all of the alkyl amino acids,
animals can synthesize only alanine and glycine; thus valine, leucine, and
isoleucine must be supplied in the diet.
Two amino acids, each containing three carbon atoms, are
derived from alanine; they are serine and cysteine. Serine
contains an alcohol group
(―CH2OH) instead of the methyl group of alanine, and cysteine contains a
mercapto group (―CH2SH). Animals can synthesize serine but not
cysteine or cystine.
Cysteine occurs in proteins predominantly in its oxidized form (oxidation in
this sense meaning the removal of hydrogen atoms), called cystine. Cystine
consists of two cysteine molecules linked by the disulfide bond (―S―S―) that
results when a hydrogen atom is removed from the mercapto group of each of the
cysteines. Disulfide bonds are important in protein structure because they
allow the linkage of two different parts of a protein molecule to—and thus the
formation of loops in—the otherwise straight chains. Some proteins contain
small amounts of cysteine with free sulfhydryl (―SH) groups.
Four amino acids, each consisting of four carbon atoms,
occur in proteins; they are aspartic acid, asparagine, threonine, and methionine.
Aspartic acid and
asparagine, which occur in large amounts, can be synthesized by
animals. Threonine and methionine cannot
be synthesized and thus are essential amino acids; i.e., they must be supplied
in the diet. Most proteins contain only small amounts of methionine.
Proteins also contain an amino acid with five carbon atoms
(glutamic acid) and a secondary amine (in proline), which is a
structure with the amino group (―NH2) bonded to the alkyl side
chain, forming a ring. Glutamic acid and
aspartic acid are dicarboxylic acids; that is, they have two carboxyl groups
(―COOH).
Glutamine is
similar to asparagine in that both are the amides of their corresponding
dicarboxylic acid forms; i.e., they have an amide group (―CONH2) in
place of the carboxyl (―COOH) of the side chain. Glutamic acid and glutamine
are abundant in most proteins; e.g., in plant proteins they sometimes comprise more
than one-third of the amino acids present. Both glutamic acid and glutamine can
be synthesized by animals.
The amino acids proline and hydroxyproline occur
in large amounts in collagen,
the protein of the connective tissue of
animals. Proline and hydroxyproline lack free amino (―NH2) groups
because the amino group is enclosed in a ring structure with the side chain;
they thus cannot exist in a zwitterion form. Although the nitrogen-containing
group (>NH) of these amino acids can form a peptide bond with the carboxyl
group of another amino acid, the bond so formed gives rise to a kink in the
peptide chain; i.e., the ring structure alters the regular bond angle of normal
peptide bonds.
Proteins usually are almost neutral molecules; that is, they
have neither acidic nor basic properties. This means that the acidic carboxyl (
―COO−) groups of aspartic and glutamic acid are about equal in
number to the amino acids with basic side chains. Three such basic amino acids,
each containing six carbon atoms, occur in proteins. The one with the simplest
structure, lysine,
is synthesized by plants but not by animals. Even some plants have a low lysine
content. Arginine is
found in all proteins; it occurs in particularly high amounts in the strongly
basic protamines (simple proteins composed of relatively few amino acids) of
fish sperm. The third basic amino acid is histidine. Both
arginine and histidine can be synthesized by animals. Histidine is a weaker
base than either lysine or arginine. The imidazole ring, a five-membered ring
structure containing two nitrogen atoms in the side chain of histidine, acts as
a buffer (i.e., a stabilizer of hydrogen ion concentration) by binding hydrogen
ions (H+) to the nitrogen atoms of the imidazole ring.
The remaining amino acids—phenylalanine, tyrosine, and tryptophan—have in
common an aromatic structure; i.e., a benzene ring is
present. These three amino acids are essential, and, while animals cannot synthesize the
benzene ring itself, they can convert phenylalanine to tyrosine.
Because these amino acids contain benzene rings, they can
absorb ultraviolet
light at wavelengths between 270 and 290 nanometres (nm; 1 nanometre =
10−9 metre = 10 angstrom units). Phenylalanine absorbs very
little ultraviolet light; tyrosine and tryptophan, however, absorb it strongly
and are responsible for the absorption band most proteins exhibit at 280–290
nanometres. This absorption is often used to determine the quantity of protein
present in protein samples.
Most proteins contain only the amino acids described above;
however, other amino acids occur in proteins in small amounts. For example, the
collagen found in connective tissue contains, in addition to hydroxyproline,
small amounts of hydroxylysine.
Other proteins contain some monomethyl-, dimethyl-, or trimethyllysine—i.e.,
lysine derivatives containing
one, two, or three methyl groups (―CH3). The amount of these unusual
amino acids in proteins, however, rarely exceeds 1 or 2 percent of the total
amino acids.
Physicochemical properties of the amino acids
The physicochemical properties of a protein are determined
by the analogous properties
of the amino acids in it.
The α-carbon atom of all amino
acids, with the exception of glycine, is asymmetric; this means that four
different chemical entities (atoms or groups of atoms) are attached to it. As a
result, each of the amino acids, except glycine, can exist in two different
spatial, or geometric, arrangements (i.e., isomers), which are
mirror images akin to right and left hands.
These isomers exhibit the property of optical rotation.
Optical rotation is the rotation of the plane of polarized light, which is
composed of light waves that vibrate in one plane, or direction, only.
Solutions of substances that rotate the plane of polarization are said to be
optically active, and the degree of rotation is called the optical rotation of
the solution.
The direction in which the light is rotated is generally designed as plus,
or d, for dextrorotatory (to the right), or as minus, or l,
for levorotatory (to the left). Some amino acids are dextrorotatory, others are
levorotatory. With the exception of a few small proteins (peptides) that occur
in bacteria, the
amino acids that occur in proteins are l-amino acids.
In bacteria, d-alanine and some other d-amino
acids have been found as components of gramicidin and bacitracin. These
peptides are toxic to
other bacteria and are used in medicine as antibiotics.
The d-alanine has also been found in some peptides of bacterial membranes.
In contrast to most organic acids and amines, the amino
acids are insoluble in organic solvents. In aqueous solutions they are dipolar
ions (zwitterions, or hybrid ions) that react with strong acids or bases in
a way that leads to the neutralization of the negatively or positively charged
ends, respectively. Because of their reactions with strong acids and strong
bases, the amino acids act as buffers—stabilizers of hydrogen ion (H+)
or hydroxide ion (OH−)
concentrations. In fact, glycine is frequently used as a buffer in the pH
range from 1 to 3 (acid solutions) and from 9 to 12 (basic solutions). In acid solutions, glycine
has a positive charge and therefore migrates to the cathode (negative
electrode of a direct-current electrical circuit with terminals in the
solution). Its charge, however, is negative in alkaline solutions, in which it
migrates to the anode (positive
electrode). At pH 6.1 glycine does not migrate, because each molecule has one
positive and one negative charge. The pH at which an amino acid does
not migrate in an electrical field is called the isoelectric point. Most of the
monoamino acids (i.e., those with only one amino group) have isoelectric points
similar to that of glycine. The isoelectric points of aspartic and glutamic
acids, however, are close to pH 3, and those of histidine, lysine, and arginine are
at pH 7.6, 9.7, and 10.8, respectively.
Amino acid sequence in protein molecules
Since each protein molecule consists of a long chain of
amino acid residues, linked to each other by peptide bonds, the
hydrolytic cleavage of all peptide bonds is a prerequisite for the quantitative
determination of the amino acid residues. Hydrolysis is
most frequently accomplished by boiling the protein with concentrated hydrochloric acid.
The quantitative determination of the amino acids is based on the discovery
that amino acids can be separated from each other by chromatography on
filter paper and made visible by spraying the paper with ninhydrin. The amino
acids of the protein hydrolysate are separated from each other by passing the
hydrolysate through a column of adsorbents, which adsorb the amino acids with
different affinities and,
on washing the column with buffer solutions, release them in a definite order.
The amount of each of the amino acids can be determined by the intensity of the
color reaction with ninhydrin.
To obtain information about the sequence of the amino acid
residues in the protein, the protein is degraded stepwise, one amino acid being
split off in each step. This is accomplished by coupling the free α-amino group
(―NH2) of the N-terminal amino acid with phenyl isothiocyanate;
subsequent mild hydrolysis does not affect the peptide bonds. The procedure,
called the Edman degradation, can be applied repeatedly; it thus reveals
the sequence of the amino acids in the peptide chain.
Unavoidable small losses that occur during each step make it
impossible to determine the sequence of more than about 30 to 50 amino acids by
this procedure. For this reason the protein is usually first hydrolyzed by
exposure to the enzyme trypsin,
which cleaves only
peptide bonds formed by the carboxyl groups of lysine and arginine. The
Edman degradation is
then applied to each of the few resulting peptides produced by the action of
trypsin. Further information can be gained by hydrolyzing another portion of
the protein with another enzyme, for instance with chymotrypsin, which splits
predominantly peptide bonds formed by the amino acids tyrosine, phenylalanine,
and tryptophan. The combination of results obtained with two or more different
proteolytic (protein degrading) enzymes was first applied by English
biochemist Frederick
Sanger, and it enabled him to elucidate the amino acid sequence of insulin. The amino acid
sequences of many other proteins subsequently were determined in the same
manner.
Levels of structural organization in proteins
Primary structure
Analytical and synthetic procedures
reveal only the primary structure of the proteins—that is, the amino acid
sequence of the peptide chains. They do not reveal information about the conformation (arrangement
in space) of the peptide chain—that is, whether the peptide chain is present as
a long straight thread or is irregularly coiled and folded into a globule.
The configuration,
or conformation, of a protein is determined by mutual attraction or repulsion
of polar or nonpolar groups in the side chains (R groups) of the
amino acids. The former have positive or negative charges in their side chains;
the latter repel water but
attract each other. Some parts of a peptide chain containing 100 to 200 amino
acids may form a loop, or helix; others may be straight or form irregular
coils.
The terms secondary, tertiary,
and quaternary structure are frequently applied to the
configuration of the peptide chain of a protein. A nomenclature committee
of the International Union of Biochemistry (IUB) has defined these terms as
follows: The primary structure of a protein is determined by its amino acid
sequence without any regard for the arrangement of the peptide chain in space.
The secondary structure is determined by the spatial arrangement of the main
peptide chain without any regard for the conformation of side chains or other
segments of the main chain. The tertiary structure is determined by both the
side chains and other adjacent segments
of the main chain, without regard for neighboring peptide chains. Finally, the
term quaternary structure is used for the arrangement of
identical or different subunits of a large protein in which each subunit is a
separate peptide chain.
Secondary structure
The nitrogen and carbon atoms
of a peptide chain
cannot lie on a straight line, because of the magnitude of the bond angles
between adjacent atoms
of the chain; the bond angle is about 110°. Each of the nitrogen and carbon
atoms can rotate to a certain extent, however, so that the chain has a limited
flexibility. Because all of the amino acids, except glycine, are
asymmetric l-amino acids, the peptide chain tends to assume an asymmetric
helical shape; some of the fibrous proteins consist of elongated helices around
a straight screw axis. Such structural features result from properties common
to all peptide chains. The product of their effects is the secondary structure
of the protein.
Tertiary structure
The tertiary structure
is the product of the interaction between the side chains (R) of the
amino acids composing the protein. Some of them contain positively or
negatively charged groups, others are polar, and still others are nonpolar. The
number of carbon atoms in the side chain varies from zero in glycine to nine in
tryptophan. Positively and negatively charged side chains have the tendency to
attract each other; side chains with identical charges repel each other. The
bonds formed by the forces between the negatively charged side chains of
aspartic or glutamic
acid on the one hand, and the positively charged side chains of lysine or arginine on
the other hand, are called salt bridges. Mutual attraction of adjacent peptide
chains also results from the formation of numerous hydrogen bonds.
Hydrogen bonds form as a result of the attraction between
the nitrogen-bound hydrogen atom (the imide
hydrogen) and the unshared pair of electrons of
the oxygen atom
in the double bonded carbon–oxygen group (the carbonyl group).
The result is a slight displacement of
the imide hydrogen toward the oxygen atom of the carbonyl group. Although the
hydrogen bond is much weaker than a covalent bond (i.e.,
the type of bond between two carbon atoms, which equally share the pair of
bonding electrons between them), the large number of imide and carbonyl groups
in peptide chains results in the formation of numerous hydrogen bonds. Another
type of attraction is that between nonpolar side chains of valine, leucine, isoleucine, and
phenylalanine; the attraction results in the displacement of water molecules and is
called hydrophobic interaction.
In proteins rich in cystine, the conformation of
the peptide chain is determined to a considerable extent by the disulfide bonds
(―S―S―) of cystine. The halves of cystine may be located in different parts of
the peptide chain and thus may form a loop closed by the disulfide bond.
If the disulfide bond is reduced (i.e., hydrogen is added)
to two sulfhydryl (―SH) groups, the tertiary structure of the protein undergoes
a drastic change—closed loops are broken and adjacent disulfide-bonded peptide
chains separate.
Quaternary structure
The nature of the quaternary structure is demonstrated by
the structure of hemoglobin.
Each molecule of human hemoglobin
consists of four peptide chains, two α-chains and two β-chains; i.e., it is a
tetramer. The four subunits are linked to each other by hydrogen bonds and
hydrophobic interaction. Because the four subunits are so closely linked, the
hemoglobin tetramer is called a molecule, even though no covalent bonds occur
between the peptide chains of the four subunits. In other proteins, the
subunits are bound to each other by covalent bonds (disulfide bridges).
The amino acid sequence
of porcine proinsulin is shown below. The arrows indicate the direction from
the N terminus of the β-chain (B) to the C terminus of the α-chain (A).
The isolation and determination of proteins
Animal material usually contains large amounts of protein
and lipids and small amounts of carbohydrate; in plants, the bulk of the dry
matter is usually carbohydrate.
If it is necessary to determine the amount of protein in a mixture of animal
foodstuffs, a sample is converted to ammonium salts by boiling with sulfuric acid and
a suitable inorganic catalyst, such
as copper sulfate (Kjeldahl
method). The method is based on the assumption that proteins contain 16
percent nitrogen,
and that nonprotein nitrogen is present in very small amounts. The assumption
is justified for most tissues from higher animals but not for insects and crustaceans, in which a
considerable portion of the body nitrogen is present in the form of chitin, a carbohydrate.
Large amounts of nonprotein nitrogen are also found in the sap of many plants.
In such cases, the precise quantitative analyses are made after the proteins
have been separated from other biological compounds.
Proteins are sensitive to heat, acids, bases, organic
solvents, and radiation exposure;
for this reason, the chemical methods employed to purify organic compounds
cannot be applied to proteins. Salts and molecules of small size are removed
from protein solutions by dialysis—i.e.,
by placing the solution into
a sac of semipermeable material, such as cellulose or
acetylcellulose, which will allow small molecules to pass through but not large
protein molecules, and immersing the sac in water or a salt
solution. Small molecules can also be removed either by passing the protein
solution through a column of resin that adsorbs
only the protein or by gel filtration. In gel filtration, the
large protein molecules pass through the column, and the small molecules are
adsorbed to the gel.
Groups of proteins are separated from each other by salting
out—i.e., the stepwise addition of sodium sulfate or
ammonium sulfate to a protein solution. Some proteins, called globulins, become
insoluble and precipitate when
the solution is half-saturated with ammonium sulfate or when its sodium sulfate
content exceeds about 12 percent. Other proteins, the albumins, can be
precipitated from the supernatant solution (i.e., the solution remaining after
a precipitation has taken place) by saturation with ammonium sulfate.
Water-soluble proteins can be obtained in a dry state by freeze-drying (lyophilization),
in which the protein solution is deep-frozen by lowering the temperature below
−15 °C (5 °F) and removing the water; the protein is obtained as a dry powder.
Most proteins are insoluble in boiling water and are
denatured by it—i.e., irreversibly converted into an insoluble material.
Heat denaturation cannot
be used with connective
tissue because the principal structural protein, collagen, is converted by
boiling water into water-soluble gelatin.
Fractionation (separation into components) of a mixture of
proteins of different molecular weight can be accomplished by gel filtration.
The size of the proteins retained by the gel depends upon the properties of the
gel. The proteins retained in the gel are removed from the column by solutions
of a suitable concentration of salts and hydrogen ions.
Many proteins were originally obtained in crystalline form,
but crystallinity is not proof of purity; many crystalline protein preparations
contain other substances. Various tests are used to determine whether a protein
preparation contains only one protein. The purity of a protein solution can be
determined by such techniques as chromatography and
gel filtration. In addition, a solution of pure protein will yield one peak
when spun in a centrifuge at
very high speeds (ultracentrifugation) and will migrate as a single band
in electrophoresis (migration
of the protein in an electrical field). After these methods and others (such
as amino acid analysis)
indicate that the protein solution is pure, it can be considered so. Because
chromatography, ultracentrifugation, and electrophoresis cannot be applied to
insoluble proteins, little is known about them; they may be mixtures of many
similar proteins.
Very small (microheterogeneous) differences in some of the
apparently pure proteins are known to occur. They are differences in the amino
acid composition of
otherwise identical proteins and are transmitted from generation to generation;
i.e., they are genetically determined. For example, some humans have two
hemoglobins, hemoglobin A
and hemoglobin S, which differ in one amino acid at a specific site in
the molecule. In
hemoglobin A the site is occupied by glutamic acid and
in hemoglobin S by valine.
Refinement of the techniques of protein analysis has resulted in the discovery
of other instances of microheterogeneity.
The quantity of a pure protein can be determined by weighing
or by measuring the ultraviolet absorbancy at 280 nanometres. The absorbency at
280 nanometres depends on the content of tyrosine and tryptophan in the
protein. Sometimes the slightly less sensitive biuret reaction, a purple color
given by alkaline protein
solutions upon the addition of copper sulfate, is used; its intensity depends
only on the number of peptide bonds
per gram, which is similar in all proteins.
Physicochemical properties of proteins
The molecular weight of
proteins
The molecular weight of
proteins cannot be determined by the methods of classical chemistry (e.g.,
freezing-point depression), because they require solutions of a higher
concentration of protein than can be prepared.
If a protein contains only one molecule of one of the amino
acids or one atom of
iron, copper, or another element, the minimum molecular weight of the protein
or a subunit can be calculated; for example, the protein myoglobin contains
0.34 gram of iron in 100 grams of protein. The atomic weight of
iron is 56; thus the minimum molecular weight of myoglobin is (56 × 100)/0.34 =
about 16,500. Direct measurements of the molecular weight of myoglobin yield the same
value. The molecular weight of hemoglobin, however, which also contains 0.34
percent iron, has been found to be 66,000 or 4 × 16,500; thus hemoglobin
contains four atoms of iron.
The method most frequently used to determine the molecular
weight of proteins is ultracentrifugation—i.e., spinning in a centrifuge at
velocities up to about 60,000 revolutions per minute. Centrifugal forces of
more than 200,000 times the gravitational force on the surface of Earth are
achieved at such velocities. The first ultracentrifuges, built in 1920, were
used to determine the molecular weight of proteins. The molecular weights of a
large number of proteins have been determined. Most consist of several
subunits, the molecular weight of which is usually less than 100,000 and
frequently ranges from 20,000 to 30,000. Proteins of very high molecular
weights are found among hemocyanins, the copper-containing respiratory proteins
of invertebrates;
some range as high as several million. Although there is no definite lower
limit for the molecular weight of proteins, short amino acid sequences are
usually called peptides.
The shape of protein molecules
Proteins
and X-ray diffractionX-ray diffraction pattern of a crystallized
enzyme.
In the technique of X-ray diffraction,
the X-rays are allowed to strike a protein crystal. The X-rays, diffracted
(bent) by the crystal, impinge on a photographic plate, forming a pattern of
spots. This method reveals that peptide chains can
assume very complicated, apparently irregular shapes. Two extremes in shape
include the closely folded structure of the globular proteins and
the elongated,
unidimensional structure of the threadlike fibrous proteins; both were
recognized many years before the technique of X-ray diffraction was developed.
Solutions of fibrous proteins are extremely viscous (i.e., sticky); those of
the globular proteins have low viscosity (i.e., they flow easily). A 5
percent solution of
a globular protein—ovalbumin, for example—easily flows through a narrow glass
tube; a 5 percent solution of gelatin, a fibrous protein,
however, does not flow through the tube, because it is liquid only
at high temperatures and solidifies at room temperature. Even solutions
containing only 1 or 2 percent of gelatin are highly viscous and flow
through a narrow tube either very slowly or only under pressure.
MacromoleculesFlow
birefringence depicting the orientation of elongated, rodlike
macromolecules (A) in resting solution, or (B) during flow through a horizontal
tube.
The elongated peptide chains of the fibrous proteins can be
imagined to become entangled not only mechanically but also by mutual
attraction of their side chains, and in this way they incorporate large amounts
of water. Most of
the hydrophilic (water-attracting) groups of the globular proteins, however,
lie on the surface of the molecules, and, as a result, globular proteins
incorporate only a few water molecules. If a solution of a fibrous protein
flows through a narrow tube, the elongated molecules become oriented parallel
to the direction of the flow, and the solution thus becomes birefringent like a
crystal; i.e., it splits a light ray into two components that travel at
different velocities and are polarized at
right angles to each other. Globular proteins do not show this phenomenon,
which is called flow birefringence. Solutions of myosin, the contractile
protein of muscles, show very high flow birefringence; other proteins with very
high flow birefringence include solutions of fibrinogen, the clotting material
of blood
plasma, and solutions of tobacco mosaic virus. The gamma-globulins
of the blood plasma show low flow birefringence, and none can be observed in
solutions of serum albumin and
ovalbumin.
Hydration of
proteins
When dry proteins are exposed to air of high water content, they
rapidly bind water up to a maximum quantity, which differs for different
proteins; usually it is 10 to 20 percent of the weight of the protein. The
hydrophilic groups of a protein are chiefly the positively charged groups in
the side chains of lysine and
arginine and the negatively charged groups of aspartic and glutamic acid.
Hydration (i.e., the binding of water) may also occur at the hydroxyl (―OH)
groups of serine and threonine or
at the amide (―CONH2) groups of asparagine and glutamine.
The binding of water molecules to either charged or polar
(partly charged) groups is explained by the dipolar structure of the water
molecule; that is, the two positively charged hydrogen atoms form an angle of
about 105°, with the negatively charged oxygen atom at the apex. The centre of the
positive charges is located between the two hydrogen atoms; the centre of the
negative charge of the oxygen atom is at the apex of the angle. The negative
pole of the dipolar water molecule binds to
positively charged groups; the positive pole binds negatively charged ones. The
negative pole of the water molecule also binds to the hydroxyl and amino groups
of the protein.
The water of hydration is essential to the structure of
protein crystals; when they are completely dehydrated, the crystalline
structure disintegrates. In some proteins this process is accompanied by denaturation and
loss of the biological function.
In aqueous solutions, proteins bind some of the water
molecules very firmly; others are either very loosely bound or form islands of
water molecules between loops of folded peptide chains.
Because the water molecules in such an island are thought to be oriented as
in ice, which is
crystalline water, the islands of water in proteins are called icebergs. Water
molecules may also form bridges between the carbonyl and imino groups of adjacent peptide
chains, resulting in structures similar to those of the pleated sheet but with
a water molecule in the position of the hydrogen bonds of that configuration. The
extent of hydration of protein molecules in aqueous solutions is important,
because some of the methods used to determine the molecular weight of
proteins yield the molecular weight of the hydrated protein. The amount of
water bound to one gram of a globular protein in solution varies
from 0.2 to 0.5 gram. Much larger amounts of water are mechanically immobilized
between the elongated peptide
chains of fibrous proteins; for example, one gram of gelatin can immobilize
at room temperature 25 to 30 grams of water.
Hydration of proteins is necessary for their solubility in
water. If the water of hydration of a protein dissolved in water is reduced by
the addition of a salt such as ammonium sulfate, the protein is no longer
soluble and is salted out, or precipitated. The salting-out process is
reversible because the protein is not denatured (i.e., irreversibly converted
to an insoluble material) by the addition of such salts as sodium chloride, sodium sulfate, or
ammonium sulfate. Some globulins, called euglobulins, are insoluble in water in
the absence of salts; their insolubility is attributed to the mutual
interaction of polar groups on the surface of adjacent molecules, a process
that results in the formation of large aggregates of
molecules. Addition of small amounts of salt causes the euglobulins to become
soluble. This process, called salting in, results from a combination
between anions (negatively
charged ions) and cations (positively
charged ions) of the salt and positively and negatively charged side chains of
the euglobulins. The combination prevents the aggregation of euglobulin
molecules by preventing the formation of salt bridges between them. The
addition of more sodium or ammonium sulfate causes the euglobulins to salt out
again and to precipitate.
Electrochemistry of proteins
Because the α-amino group and α-carboxyl group of amino
acids are converted into peptide bonds in the protein molecule, there is only
one α-amino group (at the N terminus) and one
α-carboxyl group (at the C terminus) in a given protein molecule. The
electrochemical character of a protein is affected very little by these two
groups. Of importance, however, are the numerous positively charged ammonium
groups (―NH3+) of lysine and arginine and the negatively
charged carboxyl groups (―COO−) of aspartic acid and
glutamic acid. In most proteins, the number of positively and negatively
charged groups varies from 10 to 20 per 100 amino acids.
Electrometric titration
Glycine
and electrometric titrationElectrometric titration of glycine.
When measured volumes of hydrochloric acid are
added to a solution of protein in salt-free water, the pH decreases in
proportion to the amount of hydrogen ions added until it
is about 4. Further addition of acid causes much less
decrease in pH because the protein acts as a buffer at pH values of 3 to 4. The
reaction that takes place in this pH range is the protonation of the carboxyl
group—i.e., the conversion of ―COO− into ―COOH. Electrometric
titration of an isoelectric protein with potassium hydroxide
causes a very slow increase in pH and a weak buffering action of the protein at
pH 7; a very strong buffering action occurs in the pH range from 9 to 10. The
buffering action at pH 7, which is caused by loss of protons (positively
charged hydrogen) from the imidazolium groups (i.e., the five-member ring
structure in the side chain) of histidine, is weak
because the histidine content of proteins is usually low. The much
stronger buffering action
at pH values from 9 to 10 is caused by the loss of protons from the hydroxyl group of
tyrosine and from the ammonium groups of lysine. Finally, protons are lost from
the guanidinium groups (i.e., the nitrogen-containing terminal portion of the
arginine side chains) of arginine at pH 12. Electrometric titrations of
proteins yield similar curves. Electrometric titration makes possible the
determination of the approximate number of carboxyl groups, ammonium groups,
histidines, and tyrosines per molecule of protein.
The positively and negatively charged side chains of
proteins cause them to behave like amino acids in an electrical field; that is,
they migrate during
electrophoresis at low pH values to the cathode (negative terminal) and at high
pH values to the anode (positive terminal). The isoelectric point, the pH value
at which the protein molecule does
not migrate, is in the range of pH 5 to 7 for many proteins. Proteins such
as lysozyme, cytochrome c, histone, and others rich
in lysine and
arginine, however, have isoelectric points in the pH range between 8 and 10.
The isoelectric point of pepsin, which contains
very few basic amino acids, is close to 1.
Free-boundary electrophoresis, the original method of
determining electrophoretic migration, has been replaced in many instances by
zone electrophoresis, in which the protein is placed in either a gel of starch, agar, or polyacrylamide or
in a porous medium
such as paper or cellulose acetate.
The migration of hemoglobin and
other colored proteins can be followed visually. Colorless proteins are made
visible after the completion of electrophoresis by staining them with a
suitable dye.
Conformation of globular proteins
Results of X-ray diffraction studies
Most knowledge concerning secondary and tertiary structure
of globular proteins has been obtained by the examination of their crystals
using X-ray
diffraction. In this technique, X-rays are allowed to strike the crystal;
the X-rays are diffracted by the crystal and impinge on a photographic plate,
forming a pattern of spots. The measured intensity of the diffraction pattern,
as recorded on a photographic film, depends particularly on the electron density of the
atoms in the protein crystal. This density is lowest in hydrogen atoms, and
they do not give a visible diffraction pattern. Although carbon, oxygen, and nitrogen atoms
yield visible diffraction patterns, they are present in such great number—about
700 or 800 per 100 amino acids—that the resolution of the structure of a
protein containing more than 100 amino acids is almost impossible. Resolution
is considerably improved by substituting into the side chains of certain amino
acids very heavy atoms, particularly those of heavy metals. Mercury ions,
for example, bind to the sulfhydryl (―SH) groups of cysteine. Platinum chloride
has been used in other proteins. In the iron-containing proteins, the
iron atom already
in the molecule is adequate.
Although the X-ray diffraction technique cannot resolve the
complete three-dimensional conformation (that
is, the secondary and tertiary structure
of the peptide chain),
complete resolution has been obtained by combination of the results of X-ray
diffraction with those of amino acid sequence
analysis. In this way the complete conformation of such proteins as myoglobin,
chymotrypsinogen, lysozyme, and ribonuclease has been resolved.
The X-ray diffraction method has revealed regular structural
arrangements in proteins; one is an extended form of antiparallel peptide
chains that are linked to each other by hydrogen bonds between the carbonyl and
imino groups. This conformation, called the pleated sheet, or β-structure, is
found in some fibrous proteins. Short strands of the β-structure have also been
detected in some globular proteins.
Protein
structureThe α-helix in the structural arrangement of a protein.
A second important structural arrangement is the α-helix; it
is formed by a sequence of amino acids wound around a
straight axis in either a right-handed or a left-handed spiral. Each turn of
the helix corresponds to a distance of 5.4 angstroms (= 0.54 nanometre) in the
direction of the screw axis and contains 3.7 amino acids. Hence, the length of
the α-helix per amino acid residue is 5.4 divided by 3.7, or 1.5 angstroms (1
angstrom = 0.1 nanometre). The stability of the α-helix is maintained by
hydrogen bonds between the carbonyl and imino groups of neighboring turns of
the helix. It was once thought, based on data from analyses of the myoglobin
molecule, more than half of which consists of α-helices, that the α-helix is
the predominant structural
element of the globular proteins; it is now known that myoglobin is exceptional
in this respect. The other globular proteins for which the structures have been
resolved by X-ray diffraction contain only small regions of α-helix. In most of
them the peptide chains are folded in an apparently random fashion, for which
the term random coil has been used. The term is misleading,
however, because the folding is not random; rather, it is dictated by the
primary structure and modified by the secondary and tertiary structures.
Lysozyme
and protein conformationThe simplified structure of lysozyme from hen's
egg white has a single peptide chain of 129 amino acids. The amino acid
residues are numbered from the terminal α group (N) to the terminal carboxyl
group (C). Circles indicate every fifth residue, and every tenth residue is
numbered. Broken lines indicate the four disulfide bridges. Alpha-helices are
visible in the ranges 25 to 35, 90 to 100, and 120 to 125.
The first proteins for which the internal structures were
completely resolved are the iron-containing proteins myoglobin and hemoglobin.
The investigation of the hydrated crystals of these proteins by Austrian-born
British biochemist Max Perutz and
British biochemist John C. Kendrew,
who won the 1962 Nobel
Prize for Chemistry for their work, revealed that the folding of the
peptide chains is so tight that most of the water is displaced
from the centre of the globular molecules. The amino acids that carry the
ammonium (―NH3+) and carboxyl (―COO−) groups
were found to be shifted to the surface of the globular molecules, and the
nonpolar amino acids were found to be concentrated in the interior.
Other approaches to the determination of protein
structure
None of the several other physical methods that have been
used to obtain information on the secondary and tertiary structure of proteins
provides as much direct information as the X-ray diffraction technique. Most of
the techniques, however, are much simpler than X-ray diffraction, which
requires, for the resolution of the structure of one protein, many years of
work and equipment such as electronic computers. Some of the simpler techniques
are based on the optical properties of proteins—refractivity, absorption
of light of
different wavelengths, rotation of the plane polarized light at different
wavelengths, and luminescence.
Spectrophotometric behavior
Spectrophotometry of
protein solutions (the measurement of the degree of absorbance of light by a
protein within a specified wavelength) is useful within the range of visible
light only with proteins that contain colored prosthetic groups (the nonprotein
components). Examples of such proteins include the red heme proteins of
the blood,
the purple pigments of the retina of the eye, green and yellow
proteins that contain bile pigments,
blue copper-containing proteins, and dark brown proteins called melanins. Peptide bonds,
because of their carbonyl groups, absorb light energy at very short wavelengths
(185–200 nanometres). The aromatic rings of phenylalanine, tyrosine, and
tryptophan, however, absorb ultraviolet
light between wavelengths of 280 and 290 nanometres. The absorbance of
ultraviolet light by tryptophan is greatest, that of tyrosine is less, and that
of phenylalanine is least. If the tyrosine or tryptophan content of the protein
is known, therefore, the concentration of the protein solution can
be determined by measuring its absorbance between 280 and 290 nanometres.
It will be recalled that the amino acids, with the exception
of glycine, exhibit optical activity (rotation of the plane of polarized light; see
above Physicochemical
properties of the amino acids). It is not surprising, therefore, that
proteins also are optically active. They are usually levorotatory (i.e., they
rotate the plane of polarization to the left) when polarized light of
wavelengths in the visible range is used. Although the specific rotation (a
function of the concentration of a protein solution and the distance the light
travels in it) of most l-amino acids varies from −30° tο +30°, the amino acid cystine has a
specific rotation of approximately −300°. Although the optical rotation of a
protein depends on all of the amino acids of which it is composed, the most
important ones are cystine and the aromatic amino acids phenylalanine,
tyrosine, and tryptophan. The contribution of the other amino acids to the
optical activity of a protein is negligibly small.
Chemical reactivity of proteins
Information on the internal structure of proteins can be
obtained with chemical methods that reveal whether certain groups are present
on the surface of the protein molecule and thus
able to react or whether they are buried inside the closely folded peptide chains and
thus are unable to react. The chemical reagents used in such investigations
must be mild ones that do not affect the structure of the protein.
The reactivity of tyrosine is of special interest. It has
been found, for example, that only three of the six tyrosines found in the
naturally occurring enzyme ribonuclease
can be iodinated (i.e., reacted to accept an iodine atom).
Enzyme-catalyzed breakdown of iodinated ribonuclease is used to identify the
peptides in which the iodinated tyrosines are present. The three tyrosines that
can be iodinated lie on the surface of ribonuclease; the others, assumed to be
inaccessible, are said to be buried in the molecule. Tyrosine can also be
identified by using other techniques—e.g., treatment with diazonium compounds or
tetranitromethane. Because the compounds formed are colored, they can easily be
detected when the protein is broken down with enzymes.
Cysteine can be detected by coupling with compounds such as
iodoacetic acid or
iodoacetamide; the reaction results in the formation of carboxymethylcysteine
or carbamidomethylcysteine, which can be detected by amino acid determination
of the peptides containing them. The imidazole groups of certain histidines can
also be located by coupling with the same reagents under different conditions.
Unfortunately, few other amino acids can be labelled without changes in the
secondary and tertiary structure of the protein.
Association of protein subunits
Many proteins with molecular weights of more than 50,000
occur in aqueous solutions as complexes: dimers, tetramers, and higher
polymers—i.e., as chains of two, four, or more repeating basic structural
units. The subunits, which are called monomers or protomers, usually are
present as an even number. Less than 10 percent of the polymers have been
found to have an odd number of monomers. The arrangement of the subunits is
thought to be regular and may be cyclic, cubic, or tetrahedral. Some of the
small proteins also contain subunits. Insulin, for example,
with a molecular
weight of about 6,000, consists of two peptide chains linked to each
other by disulfide bridges (―S―S―). Similar interchain disulfide bonds have
been found in the immunoglobulins. In other proteins, hydrogen bonds and
hydrophobic bonds (resulting from the interaction between the amino acid side
chains of valine, leucine, isoleucine, and
phenylalanine) cause the formation of aggregates of
the subunits. The subunits of some proteins are identical; those of others
differ. Hemoglobin is a tetramer consisting of two α-chains and two β-chains.
Protein denaturation
When a solution of
a protein is boiled, the protein frequently becomes insoluble—i.e., it is
denatured—and remains insoluble even when the solution is cooled. The
denaturation of the proteins of egg white by heat—as when boiling an egg—is an
example of irreversible denaturation. The denatured protein has the same
primary structure as the original, or native, protein. The weak forces between
charged groups and the weaker forces of mutual attraction of nonpolar groups
are disrupted at elevated temperatures, however; as a result, the tertiary
structure of the protein is lost. In some instances the original structure of
the protein can be regenerated; the process is called renaturation.
Denaturation can be brought about in various ways. Proteins
are denatured by treatment with alkaline or acid, oxidizing or reducing
agents, and certain organic solvents.
Interesting among denaturing agents are those that affect the secondary and
tertiary structure without affecting the primary structure. The agents most
frequently used for this purpose are urea and guanidinium
chloride. These molecules, because of their high affinity for peptide bonds, break
the hydrogen bonds and the salt bridges between positive and negative side
chains, thereby abolishing the tertiary structure of the peptide chain. When
denaturing agents are removed from a protein solution, the native protein
re-forms in many cases. Denaturation can also be accomplished by reduction of
the disulfide bonds of cystine—i.e., conversion of the disulfide bond (―S―S―)
to two sulfhydryl groups (―SH). This, of course, results in the formation of
two cysteines. Reoxidation of the cysteines by exposure to air sometimes
regenerates the native protein. In other cases, however, the wrong cysteines
become bound to each other, resulting in a different protein. Finally,
denaturation can also be accomplished by exposing proteins to organic solvents such
as ethanol or acetone. It is believed
that the organic solvents interfere
with the mutual attraction of nonpolar groups.
Some of the smaller proteins, however, are extremely stable,
even against heat; for example, solutions of ribonuclease can be exposed for
short periods of time to temperatures of 90 °C (194 °F) without undergoing
significant denaturation. Denaturation does not involve identical changes in
protein molecules. A common property of denatured proteins, however, is the
loss of biological activity—e.g., the ability to act as enzymes or hormones.
Although denaturation had long been considered an
all-or-none reaction, it is now thought that many intermediary states exist
between native and denatured protein. In some instances, however, the breaking
of a key bond could be followed by the complete breakdown of the conformation of
the native protein.
Although many native proteins are resistant to the action of
the enzyme trypsin,
which breaks down proteins during digestion, they
are hydrolyzed by the same enzyme after denaturation. The peptide bonds that
can be split by trypsin are inaccessible in the native proteins but become
accessible during denaturation. Similarly, denatured proteins give more intense
color reactions for tyrosine, histidine, and arginine
than do the same proteins in the native
state. The increased accessibility of reactive groups of denatured proteins
is attributed to
an unfolding of the peptide chains.
If denaturation can be brought about easily and if
renaturation is difficult, how is the native conformation of globular proteins
maintained in living organisms, in which they are produced stepwise, by
incorporation of one amino acid at a
time? Experiments on the biosynthesis of
proteins from amino acids containing radioactive carbon or heavy hydrogen reveal
that the protein molecule grows
stepwise from the N terminus
to the C terminus; in each step a single amino acid residue is incorporated. As
soon as the growing peptide chain contains six or seven amino acid residues,
the side chains interact with each other and thus cause deviations from
the straight or β-chain configuration.
Depending on the nature of the side chains, this may result in the formation of
an α-helix or of loops closed by hydrogen bonds or disulfide bridges. The final
conformation is probably frozen when the peptide chain attains a length of 50
or more amino acid residues.
Conformation of
proteins in interfaces
Like many other substances with both hydrophilic and
hydrophobic groups, soluble proteins tend to migrate into the interface between
air and water or oil and water; the term oil here means a
hydrophobic liquid such
as benzene or xylene. Within the
interface, proteins spread, forming thin films. Measurements of the surface tension,
or interfacial tension,
of such films indicate that tension is reduced by the protein film. Proteins,
when forming an interfacial film, are present as a monomolecular layer—i.e., a
layer one molecule in
height. Although it was once thought that globular protein molecules unfold
completely in the interface, it has now been established that many proteins can
be recovered from films in the native
state. The application of lateral pressure on a protein film causes it to
increase in thickness and finally to form a layer with a height corresponding
to the diameter of the native protein molecule. Protein molecules in an
interface, because of Brownian motions (molecular
vibrations), occupy much more space than do those in the film after the
application of pressure. The Brownian motion of compressed molecules is limited
to the two dimensions of the interface, since the protein molecules cannot move
upward or downward.
The motion of protein molecules at the air–water interface
has been used to determine the molecular weight of
proteins. The technique involves measuring the force exerted by the protein
layer on a barrier.
When a protein solution is
vigorously shaken in air, it forms a foam, because the soluble
proteins migrate into the air–water interface and persist there, preventing or
slowing the reconversion of the foam into a homogeneous solution.
Some of the unstable, easily modified proteins are denatured when spread in the
air–water interface. The formation of a permanent foam when egg white is
vigorously stirred is an example of irreversible denaturation by
spreading in a surface.
Classification of proteins
Classification by solubility
CollagenA
three-dimensional model of a collagen molecule.
After two German chemists, Emil Fischer and Franz
Hofmeister, independently stated in 1902 that proteins are essentially
polypeptides consisting of many amino acids, an
attempt was made to classify proteins according to their chemical and physical
properties, because the biological function of proteins had not yet been
established. (The protein character of enzymes was not
proved until the 1920s.) Proteins were classified primarily according to their
solubility in a number of solvents. This
classification is no longer satisfactory, however, because proteins of quite
different structure and function sometimes have similar solubilities;
conversely, proteins of the same function and similar structure sometimes have
different solubilities. The terms associated with the old classification,
however, are still widely used. They are defined below.
Keratin Scanning
electron micrograph showing strands of keratin in a feather, magnified 186×.
Albumins are
proteins that are soluble in water and in water
half-saturated with ammonium sulfate. On the other hand, globulins are
salted out (i.e., precipitated) by half-saturation with ammonium sulfate.
Globulins that are soluble in salt-free water are called pseudoglobulins; those
insoluble in salt-free water are euglobulins. Both prolamins and glutelins,
which are plant proteins,
are insoluble in water; the prolamins dissolve in 50
to 80 percent ethanol,
the glutelins in acidified or alkaline solution. The term protamine is
used for a number of proteins in fish sperm that consist of approximately 80
percent arginine and
therefore are strongly alkaline.
Histones,
which are less alkaline, apparently occur only in cell nuclei, where
they are bound to nucleic
acids. The term scleroproteins has
been used for the insoluble proteins of animal organs. They include keratin, the insoluble
protein of certain epithelial tissues such as the skin or hair, and collagen, the protein of
the connective
tissue. A large group of proteins has been called conjugated
proteins, because they are complex molecules of protein consisting of
protein and nonprotein moieties. The nonprotein portion is called the
prosthetic group. Conjugated proteins
can be subdivided into mucoproteins, which, in addition to protein, contain
carbohydrate; lipoproteins,
which contain lipids; phosphoproteins, which are rich in phosphate;
chromoproteins, which contain pigments such as iron-porphyrins, carotenoids, bile
pigments, and melanin;
and finally, nucleoproteins, which contain nucleic acid.
The weakness of the above classification lies in the fact
that many, if not all, globulins contain small amounts of carbohydrate; thus
there is no sharp borderline between globulins and mucoproteins. Moreover, the
phosphoproteins do not have a prosthetic group that can be isolated; they are
merely proteins in which some of the hydroxyl groups of serine are
phosphorylated (i.e., contain phosphate). Finally, the globulins include
proteins with quite different roles—enzymes, antibodies, fibrous
proteins, and contractile proteins.
Classification by biological functions
In view of the unsatisfactory state of the old
classification, it is preferable to classify the proteins according to their
biological function. Such a classification is far from ideal, however, because
one protein can have more than one function. The contractile protein myosin, for example, also
acts as an ATPase (adenosine triphosphatase), an enzyme that
hydrolyzes adenosine
triphosphate (removes a phosphate group from ATP by introducing a
water molecule). Another problem with functional classification is that the
definite function of a protein frequently is not known. A protein cannot be
called an enzyme as long as its substrate (the
specific compound upon
which it acts) is not known. It cannot even be tested for its enzymatic action
when its substrate is not known.
Special structure and function of proteins
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Despite its weaknesses, a functional classification is used
here in order to demonstrate, whenever possible, the correlation between the
structure and function of a protein. The structural, fibrous proteins are
presented first, because their structure is simpler than that of the globular
proteins and more clearly related to their function, which is the maintenance
of either a rigid or a flexible structure.
Structural proteins
Collagenous
fibersRandomly oriented collagenous fibers of varying size in a thin
spread of loose areolar connective tissue (magnified about 370 ×).
Collagen is the structural protein of bones, tendons, ligaments, and skin. For many years
collagen was considered to be insoluble in water. Part of the collagen
of calf skin, however, can be extracted with citrate buffer at pH 3.7. A precursor of
collagen called procollagen is converted in the body into collagen. Procollagen
has a molecular
weight of 120,000. Cleavage of one or a few peptide bonds of
procollagen yields collagen, which has three subunits, each with a molecular
weight of 95,000; therefore, the molecular weight of collagen is 285,000 (3 ×
95,000). The three subunits are wound as spirals around an elongated straight
axis. The length of each subunit is 2,900 angstroms, and its
diameter is approximately 15 angstroms. The three chains are staggered, so that
the trimer has no definite terminal limits.
Collagen differs from all other proteins in its high content
of proline and hydroxyproline.
Hydroxyproline does not occur in significant amounts in any other protein
except elastin. Most of the proline in collagen is present in the
sequence glycine–proline-X,
in which X is frequently alanine or
hydroxyproline. Collagen does not contain cystine or tryptophan and
therefore cannot substitute for other proteins in the diet. The presence of
proline causes kinks in the peptide chain and thus reduces the length of
the amino acid unit
from 3.7 angstroms in the extended chain of the β-structure to 2.86 angstroms
in the collagen chain. In the intertwined triple
helix, the glycines are inside, close to the axis; the prolines are outside.
Native collagen resists the action of trypsin but is
hydrolyzed by the bacterial enzyme collagenase.
When collagen is boiled with water, the triple helix is destroyed, and the
subunits are partially hydrolyzed; the product is gelatin. The unfolded
peptide chains of gelatin trap large amounts of water, resulting in a
hydrated molecule.
When collagen is treated with tannic acid or with
chromium salts, cross
links form between the collagen fibers, and it becomes insoluble; the
conversion of hide into leather is based on this tanning process. The tanned
material is insoluble in hot water and cannot be converted to
gelatin. On exposure to water at 62° to 63° C (144° to 145° F), however, the
cross links formed by the tanning agents collapse, and the leather contracts
irreversibly to about one-third its original volume.
Collagen seems to undergo an aging process in living
organisms that may be caused by the formation of cross links between collagen
fibers. They are formed by the conversion of some lysine side chains to
aldehydes (compounds with the general structure RCHO), and the
combination of the aldehydes with
the ε-amino groups of intact lysine side chains. The protein elastin, which occurs in
the elastic fibers of connective tissue,
contains similar cross links and may result from the combination of collagen
fibers with other proteins. When cross-linked collagen or elastin is degraded, products of
the cross-linked lysine fragments,
called desmosins and isodesmosins, are formed.
Keratin, the structural protein of epithelial cells in the
outermost layers of the skin, has been isolated from hair, nails, hoofs, and
feathers. Keratin is completely insoluble in cold or hot water; it is not
attacked by proteolytic
enzymes (i.e., enzymes that break apart, or lyse, protein molecules),
and therefore cannot replace proteins in the diet. The great stability of
keratin results from the numerous disulfide bonds of cystine. The amino acid composition of
keratin differs from that of collagen. Cystine may
account for 24 percent of the total amino acids. The peptide chains of
keratin are arranged in approximately equal amounts of antiparallel and
parallel pleated sheets, in which the peptide chains are linked to each other
by hydrogen
bonds between the carbonyl and imino groups.
Reduction of the disulfide bonds to sulfhydryl groups
results in dissociation of the peptide chains, the molecular weight of
which is 25,000 to 28,000 each. The formation of permanent waves in the
beauty treatment of
hair is based on partial reduction of the disulfide bonds of hair keratin by
thioglycol, or some other mild reducing agent, and subsequent oxidation of the
sulfhydryl groups (―SH) in the reoriented hair to disulfide bonds (―S―S―) by
exposure to the oxygen of
the air.
The length of keratin fibers depends on their water content. They
can bind approximately 16 percent of water; this hydration is accompanied by an
increase in the length of the fibers of 10 to 12 percent.
The most thoroughly investigated keratin is hair keratin,
particularly that of wool.
It consists of a mixture of peptides with high and low cystine content. When
wool is heated in water to about 90° C (190° F), it shrinks irreversibly. This
is attributed to the breakage of hydrogen bonds and other noncovalent bonds;
disulfide bonds do not seem to be affected.
Others
The most thoroughly investigated scleroprotein has
been fibroin, the
insoluble material of silk.
The raw silk comprising the cocoon of the
silkworm consists of two proteins. One, sericin, is soluble in hot water; the
other, fibroin, is not. The amino acid composition of the latter differs from
that of all other proteins. It contains large amounts of glycine, alanine, tyrosine, and serine; small amounts of
the other amino acids; and no sulfur-containing ones. The peptide chains are
arranged in antiparallel β-structures. Fibroin is partly soluble in
concentrated solutions of lithium thiocyanate or in mixtures of cupric salts
and ethylene diamine. Such solutions contain a protein of molecular weight
170,000, which is a dimer of two subunits.
Little is known about either the scleroproteins of the
marine sponges or
the insoluble proteins of the cellular membranes of animal cells. Some of the
membranes are soluble in detergents; others,
however, are detergent-insoluble.
The muscle proteins
The total amount of muscle proteins in
mammals, including humans, exceeds that of any other protein. About 40 percent
of the body weight of a healthy human adult
weighing about 70 kilograms (150 pounds) is muscle, which is composed of about
20 percent muscle protein. Thus, the human body contains
about 5 to 6 kilograms (11 to 13 pounds) of muscle protein. An albumin-like
fraction of these proteins, originally called myogen, contains various
enzymes—phosphorylase, aldolase, glyceraldehyde phosphate dehydrogenase, and
others; it does not seem to be involved in contraction. The globulin fraction
contains myosin, the contractile protein, which also occurs in blood platelets, small bodies
found in blood. Similar contractile substances occur in other contractile
structures; for example, in the cilia or flagella (whiplike
organs of locomotion) of bacteria and protozoans. In contrast
to the scleroproteins, the contractile proteins are soluble in salt solutions
and susceptible to
enzymatic digestion.
The energy required for muscle contraction is provided by
the oxidation of carbohydrates or lipids. The term mechanochemical
reaction has been used for this conversion of chemical into mechanical energy.
The molecular process underlying the reaction is known to involve the fibrous
muscle proteins, the peptide chains
of which undergo a change in conformation during
contraction.
Myosin,
which can be removed from fresh muscle by adding it to a chilled solution of
dilute potassium
chloride and sodium bicarbonate,
is insoluble in water.
Myosin, solutions of which are highly viscous, consists of an
elongated—probably double-stranded—peptide chain, which is coiled at both ends
in such a way that a terminal globule
is formed. The length of the molecule is
approximately 160 nanometres and its average diameter 2.6 nanometres. The
equivalent weight of each of the two terminal globules is approximately 30,000;
the molecular
weight of myosin is close to 500,000. Trypsin splits myosin into large
fragments called meromyosin. Myosin contains many amino acids with positively
and negatively charged side chains; they form 18 and 16 percent, respectively,
of the total number of amino acids. Myosin catalyzes the hydrolytic cleavage
of ATP (adenosine
triphosphate). A smaller protein with properties similar to those of myosin is
tropomyosin. It has a molecular weight of 70,000 and dimensions of
45 by 2 nanometres. More than 90 percent of its peptide chains are present in
the α-helix form.
The
structure of actin and myosin filamentsMyosin proteins bind to actin
filaments and use ATP hydrolysis to drive contraction and movement, acting as a
molecular motor in muscles and nonmuscle cells.
Myosin combines easily with another muscle protein
called actin, the
molecular weight of which is about 50,000; it forms 12 to 15 percent of the
muscle proteins. Actin can exist in two forms—one, G-actin, is globular; the
other, F-actin, is fibrous. Actomyosin is a complex molecule formed by one
molecule of myosin and one or two molecules of actin. In muscle, actin and
myosin filaments are oriented parallel to each other and to the long axis of
the muscle. The actin filaments are linked to each other lengthwise by fine
threads called S filaments. During contraction the
S filaments shorten, so that the actin filaments slide toward each other, past
the myosin filaments, thus causing a shortening of the muscle (for a detailed
description of the process, see muscle:
Striated muscle).
Fibrinogen and
fibrin
Fibrin
in blood clottingRed blood cells (erythrocytes) trapped in a mesh of
fibrin threads. Fibrin, a tough, insoluble protein formed after injury to the
blood vessels, is an essential component of blood clots.
Fibrinogen, the protein of the blood plasma, is converted
into the insoluble protein fibrin during the
clotting process. The fibrinogen-free fluid obtained after removal of the clot,
called blood serum,
is blood plasma minus
fibrinogen. The fibrinogen content of the blood plasma is 0.2 to 0.4 percent.
Fibrinogen can be precipitated from the blood plasma by
half-saturation with sodium
chloride. Fibrinogen solutions are highly viscous and show strong flow
birefringence. In electron micrographs the molecules appear as rods with a
length of 47.5 nanometres and a diameter of 1.5
nanometres; in addition, two terminal and a central nodule are visible. The
molecular weight is 340,000. An unusually high percentage, about 36 percent, of
the amino acid side
chains are positively or negatively charged.
The clotting process is initiated by the enzyme thrombin,
which catalyzes the breakage of a few peptide bonds of fibrinogen; as a result,
two small fibrinopeptides with molecular weights of 1,900 and 2,400 are
released. The remainder of the fibrinogen molecule, a monomer, is soluble and
stable at pH values less than 6 (i.e., in acid solutions). In
neutral solution (pH 7) the monomer is converted into a larger molecule,
insoluble fibrin; this results from the formation of new peptide bonds. The
newly formed peptide bonds form intermolecular and intramolecular cross links,
thus giving rise to a large clot, in which all molecules are linked to each
other. Clotting, which takes place only in the presence of calcium ions, can be
prevented by compounds such
as oxalate or citrate, which have a high affinity for
calcium ions.
Albumins, globulins, and other soluble proteins
The blood plasma, the lymph, and other animal
fluids usually contain one to seven grams of protein per 100 millilitres of
fluid, which includes small amounts of hundreds of enzymes and a large
number of protein hormones.
The discussion below is limited largely to the proteins that occur in large
amounts and can be easily isolated from the body fluids.
Proteins of the blood serum
Human blood serum contains about 7
percent protein, two-thirds of which is in the albumin fraction;
the other third is in the globulin fraction. Electrophoresis of serum reveals a
large albumin peak and three smaller globulin peaks, the
alpha-, beta-, and gamma-globulins. The amounts of alpha-, beta-, and
gamma-globulin in normal human serum are
approximately 1.5, 1.9, and 1.1 percent, respectively. Each globulin fraction
is a mixture of many different proteins, as has been demonstrated by
immunoelectrophoresis. In this method, serum from an animal (e.g., a rabbit)
injected with human serum is allowed to diffuse into the four protein
bands—albumin, alpha-, beta-, and gamma-globulin—obtained from the
electrophoresis of human serum. Because the animal has previously been injected
with human serum, its blood contains antibodies (substances
formed in response to a foreign substance introduced into the body) against
each of the human serum proteins; each antibody combines
with the serum protein (antigen)
that caused its formation in the animal. The result is the formation of about
20 regions of insoluble antigen-antibody precipitate, which appear as white
arcs in the transparent gel of
the electrophoresis medium. Each region corresponds to a different human serum
protein.
Serum
albumin is much less heterogeneous (i.e.,
contains fewer distinct proteins) than are the globulins; in fact, it is one of
the few serum proteins that can be obtained in a crystalline form. Serum
albumin combines easily with many acidic dyes (e.g., Congo red and
methyl orange); with bilirubin,
the yellow bile pigment; and with fatty acids. It seems
to act, in living organisms, as a carrier for certain biological substances.
Present in blood serum in relatively high concentration, serum albumin also
acts as a protective colloid,
a protein that stabilizes other proteins. Albumin (molecular weight of 68,000)
has a single free sulfhydryl (―SH) group, which on oxidation forms a disulfide
bond with the sulfhydryl group of another serum albumin molecule, thus forming a
dimer. The isoelectric point of serum albumin is pH 4.7.
The alpha-globulin fraction of blood serum is a mixture of
several conjugated
proteins. The best known are an α-lipoprotein (combination
of lipid and
protein) and two mucoproteins (combinations of carbohydrate and
protein). One mucoprotein is called orosomucoid, or α1-acid
glycoprotein; the other is called haptoglobin because
it combines specifically with globin, the protein component of hemoglobin.
Haptoglobin contains about 20 percent carbohydrate. The beta-globulin fraction
of serum contains, in addition to lipoproteins and mucoproteins, two
metal-binding proteins, transferrin and
ceruloplasmin, which bind iron and copper, respectively. They
are the principal iron and copper carriers of the blood.
Antibody
structureThe four-chain structure of an antibody, or immunoglobulin,
molecule. The basic unit is composed of two identical light (L) chains and two
identical heavy (H) chains, which are held together by disulfide bonds to form
a flexible Y shape. Each chain is composed of a variable (V) region and a
constant (C) region.
The gamma-globulins are the most heterogeneous globulins.
Although most have a molecular weight of
approximately 150,000, that of some, called macroglobulins, is as high as
800,000. Because typical antibodies are of the same size and exhibit the same
electrophoretic behavior as γ-globulins, they are called immunoglobulins.
The designation IgM
or gamma M (γM) is used for the macroglobulins; the designation IgG or gamma G
(γG) is used for γ−globulins of molecular weight 150,000.
Milk proteins
Milk contains the following: an albumin, α-lactalbumin; a
globulin, beta-lactoglobulin; and a phosphoprotein, casein. If acid is added to milk,
casein precipitates. The remaining watery liquid (the
supernatant solution), or whey,
contains α-lactalbumin and β-lactoglobulin. Both have been obtained in
crystalline form; in bovine milk, their molecular weights are approximately
14,000 and 18,400, respectively. Lactoglobulin also occurs as a dimer of
molecular weight 37,000. Genetic variations can produce small variations in
the amino acid composition of
lactoglobulin. The amino acid composition and the tertiary structure of
lactalbumin resemble that of lysozyme, an egg
protein.
Casein is
precipitated not only by the addition of acid but also by the action of
the enzyme rennin, which is found in
gastric juice. Rennin from calf stomachs is used to precipitate casein, from
which cheese is
made. Milk fat precipitates with casein; milk sugar, however,
remains in the supernatant (whey). Casein is a mixture of several similar
phosphoproteins, called α-, β-, γ−, and κ-casein, all of which contain some
serine side chains combined with phosphoric acid.
Approximately 75 percent of casein is α-casein. Cystine has been
found only in κ-casein. In milk, casein seems to form polymeric globules
(micelles) with radially arranged monomers, each with a
molecular weight of 24,000; the acidic side chains occur predominantly on the
surface of the micelle,
rather than inside.
Egg proteins
About 50 percent of the proteins of egg white are
composed of ovalbumin, which is easily obtained in crystals. Its molecular
weight is 46,000 and its amino acid composition differs from that of serum
albumin. Other proteins of egg white are conalbumin, lysozyme, ovoglobulin,
ovomucoid, and avidin. Lysozyme is an enzyme that hydrolyzes the carbohydrates
found in the capsules certain bacteria secrete around
themselves; it causes lysis (disintegration) of the bacteria. The molecular
weight of lysozyme is 14,100. Its three-dimensional structure is similar to
that of α-lactalbumin, which stimulates the formation of lactose by the enzyme
lactose synthetase. Lysozyme has also been found in the urine of patients
suffering from leukemia, meningitis, and renal
disease.
Avidin is
a glycoprotein that combines specifically with biotin, a vitamin. In animals fed
large amounts of raw egg white, the action of avidin results in “egg-white
injury.” The molecular weight of avidin, which forms a tetramer, is 16,200. Its
amino acid sequence is known.
Egg-yolk proteins contain a mixture of lipoproteins and
livetins. The latter are similar to serum albumin, α-globulin, and β-globulin.
The yolk also contains a phosphoprotein, phosvitin. Phosvitin, which has also
been found in fish sperm, has a molecular weight of 40,000 and an unusual amino
acid composition; one third of its amino acids are phosphoserine.
Protamines and
histones
Protamines are
found in the sperm cells of fish. The most thoroughly investigated protamines
are salmine from salmon sperm
and clupeine from herring sperm.
The protamines are bound to deoxyribonucleic acid (DNA), forming
nucleoprotamines. The amino acid composition of the protamines is simple; they
contain, in addition to large amounts of arginine, small amounts
of five or six other amino acids. The composition of
the salmine molecule, for example, is: Arg51, Ala4, Val4,
Ile1, Pro7, and Ser6, in which the subscript
numbers indicate the number of each amino acid in the molecule. Because of the
high arginine content, the isoelectric points of the protamines are at pH
values of 11 to 12; i.e., the protamines are alkaline. The molecular weights of
salmine and clupeine are close to 6,000. All of the protamines investigated
thus far are mixtures of several similar proteins.
The histones are
less basic than the protamines. They contain high amounts of either lysine or arginine
and small amounts of aspartic acid and glutamic acid.
Histones occur in combination with DNA as nucleohistones in the nuclei of the
body cells of animals and plants, but not in animal sperm. The molecular
weights of histones vary from 10,000 to 22,000. In contrast to the protamines,
the histones contain most of the 20 amino acids, with the exception of tryptophan and
the sulfur-containing ones. Like the protamines, histone preparations
are heterogeneous mixtures.
The amino acid sequence of some of the histones has been determined.
Plant proteins
Plant proteins, mostly globulins, have been obtained chiefly
from the protein-rich seeds of cereals and legumes. Small amounts of
albumins are found in seeds.
The best known globulins, insoluble in water, can be extracted
from seeds by treatment with 2 to 10 percent solutions of sodium chloride. Many plant
globulins have been obtained in crystalline form; they include edestin
from hemp, molecular weight 310,000;
amandin from almonds,
330,000; concanavalin A (42,000) and B (96,000); and canavalin (113,000) from
jack beans. They are polymers of smaller subunits; edestin, for example, is a
hexamer of a subunit with a molecular weight of 50,000, and concanavalin B a
trimer of a subunit with a molecular weight of 30,000. After extraction of
lipids from cereal seeds by ether and alcohol, further
extraction with water containing 50 to 80 percent of alcohol yields proteins
that are insoluble in water but soluble in water–ethanol mixtures and have been
called prolamins.
Their solubility in aqueous ethanol may result from their high proline and glutamine content. Gliadin, the prolamin from wheat, contains 14 grams of
proline and 46 grams of glutamic acid in
100 grams of protein; most of the glutamic acid is in the form of glutamine.
The total amounts of the basic amino acids (arginine, lysine, and histidine) in gliadin
are only 5 percent of the weight of gliadin. Because the glysine content is
either low or nonexistent, human populations
dependent on grain as a sole protein source suffer from lysine deficiency.
Combination of proteins with prosthetic groups
The link between a protein molecule and its
prosthetic group is a covalent bond (an electron-sharing bond)
in the glycoproteins, the biliproteins, and some of the heme proteins. In lipoproteins, nucleoproteins, and
some heme proteins, the two components are linked by noncovalent bonds; the
bonding results from the same forces that are responsible for the tertiary structure
of proteins: hydrogen
bonds, salt bridges between positively and negatively charged groups,
disulfide bonds, and mutual interaction of hydrophobic groups. In the
metalloproteins (proteins with a metal element as a prosthetic group),
the metal ion usually
forms a centre to which various groups are bound.
Some of the conjugated proteins have been mentioned in
preceding sections because they occur in the blood serum,
in milk, and in eggs; others are discussed below in sections dealing with
respiratory proteins and enzymes.
Mucoproteins and glycoproteins
The prosthetic groups in mucoproteins and glycoproteins
are oligosaccharides (carbohydrates
consisting of a small number of simple sugar molecules)
usually containing from four to 12 sugar molecules; the most common sugars
are galactose,
mannose, glucosamine, and galactosamine. Xylose, fucose, glucuronic acid, sialic acid, and other
simple sugars sometimes also occur. Some mucoproteins contain 20 percent or
more of carbohydrate,
usually in several oligosaccharides attached to different parts of the peptide chain.
The designation mucoprotein
is used for proteins with more than 3 to 4 percent carbohydrate; if the
carbohydrate content is less than 3 percent, the protein is sometimes called a
glycoprotein or simply a protein.
Mucoproteins, highly viscous proteins originally called
mucins, are found in saliva,
in gastric juice, and in other animal secretions. Mucoproteins occur in large
amounts in cartilage,
synovial fluid (the lubricating fluid of joints and tendons), and egg white.
The mucoprotein of cartilage is formed by the combination of collagen with
chondroitinsulfuric acid, which is a polymer of either
glucuronic or iduronic acid and acetylhexosamine or acetylgalactosamine. It is
not yet clear
whether or not chondroitinsulfate is bound to collagen by covalent bonds.
Lipoproteins and proteolipids
The bond between the protein and the lipid portion of
lipoproteins and proteolipids is a noncovalent one. It is thought that some of
the lipid is enclosed in a meshlike arrangement of peptide chains and becomes
accessible for reaction only after the unfolding of the chains by denaturing agents.
Although lipoproteins in the α- and β-globulin fraction of blood serum are
soluble in water (but insoluble in organic solvents), some of the brain lipoproteins,
because they have a high lipid content, are soluble in organic solvents; they
are called proteolipids. The β-lipoprotein of human blood serum is a
macroglobulin with a molecular weight of about 1,300,000, 70 percent of which
is lipid; of the lipid, about 30 percent is phospholipid and 40 percent cholesterol and compounds derived
from it. Because of their lipid content, the lipoproteins have the lowest
density (mass per unit volume) of all proteins and are usually classified as
low- and high-density lipoproteins (LDL and HDL).
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Colored lipoproteins are formed by the combination of
protein with carotenoids.
Crustacyanin, the pigment of lobsters, crayfish, and other crustaceans,
contains astaxanthin, which is a compound derived
from carotene.
Among the most interesting of the colored lipoproteins are the pigments of
the retina of
the eye. They
contain retinal, which is a compound derived from carotene and which is formed
by the oxidation of vitamin
A. In rhodopsin,
the red pigment of the retina, the aldehyde group
(―CHO) of retinal forms a covalent bond with an amino (―NH2) group
of opsin, the protein carrier. Color vision is mediated by the presence of
several visual pigments in the retina that differ from rhodopsin either in the
structure of retinal or in that of the protein carrier.
Metalloproteins
Proteins in which heavy metal ions are bound
directly to some of the side chains of histidine, cysteine, or some
other amino acid are
called metalloproteins. Two metalloproteins, transferrin and
ceruloplasmin, occur in the globulin fractions
of blood serum; they act as carriers of iron and copper, respectively.
Transferrin has a molecular weight of about 80,000 and consists of two
identical subunits, each of which contains one ferric ion (Fe3+)
that seems to be bound to tyrosine. Several
genetic variants of transferrin are known to occur in humans. Another iron
protein, ferritin, which contains 20 to 22 percent iron, is the form in which
iron is stored in animals; it has been obtained in crystalline form from liver and spleen. A molecule
consisting of 20 subunits, its molecular weight is approximately 480,000. The
iron can be removed by reduction from the ferric (Fe3+) to the
ferrous (Fe2+) state. The iron-free protein, apoferritin, is synthesized in
the body before the iron is incorporated.
Green plants and some photosynthetic and nitrogen-fixing
bacteria (i.e., bacteria that
convert atmospheric nitrogen,
N2, into amino acids and proteins) contain various ferredoxins. They
are small proteins containing 50 to 100 amino acids and a chain of iron and
disulfide units (FeS2), in which some of the sulfur atoms are
contributed by cysteine; others are sulfide ions (S2−). The number
of FeS2 units per ferredoxin molecule varies from five in the
ferredoxin of spinach to 10 in the ferredoxin of certain bacteria. Ferredoxins
act as electron carriers in photosynthesis and
in nitrogen
fixation.
Ceruloplasmin is
a copper-containing globulin that has a molecular weight of 151,000; the
molecule consists of eight subunits, each containing one copper ion. Ceruloplasmin is the
principal carrier of copper in organisms, although copper can also be
transported by the iron-containing globulin transferrin. Another
copper-containing protein, copper-zinc superoxide
dismutase (formerly known as erythrocuprein), has been isolated from red blood cells;
it has also been found in the liver and in the brain. The molecule, which
consists of two subunits of similar size, contains copper ions and zinc ions.
Because of their copper content, ceruloplasmin and copper-zinc superoxide
dismutase possess catalytic activity in oxidation-reduction
reactions.
Many animal enzymes contain zinc ions, which are usually
bound to the sulfur of cysteine. Horse kidneys contain the protein
metallothionein, which contain zinc and cadmium; both are bound
to sulfur. A vanadium-protein
complex (hemovanadin) has been found in surprisingly high amounts in
yellowish-green cells (vanadocytes) of tunicates, which are
marine invertebrates.
Heme proteins
and other chromoproteins
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Although the heme proteins contain iron, they are usually
not classified as metalloproteins, because their prosthetic group is an iron-porphyrin complex
in which the iron is bound very firmly. The intense red or brown color of the
heme proteins is not caused by iron but by porphyrin, a complex cyclic
structure. All porphyrin compounds absorb light intensely at or close to 410
nanometres. Porphyrin consists of four pyrrole rings (five-membered closed
structures containing one nitrogen and four carbon atoms)
linked to each other by methine groups (―CH=). The iron atom is kept in the
centre of the porphyrin ring by interaction with the four nitrogen atoms. The
iron atom can combine with two other substituents; in oxyhemoglobin, one
substituent is a histidine of the protein carrier, the other is an oxygen molecule. In
some heme proteins, the protein is also bound covalently to the side chains of
porphyrin. Heme proteins are described below (see Respiratory
proteins).
The chromoprotein melanin, a pigment found
in dark skin, dark hair, and melanotic tumors, occurs in every
major group of living organisms and appears to be remarkably diverse in
structure. In humans, melanin produced by melanocytes may
be dark brown (eumelanin) or pale red or yellowish (phaeomelanin). The
different types are synthesized via different pathways, though they share the
same initial step—the oxidation of tyrosine.
Blue-green
algaeThe Morning Glory Pool at Yellowstone National Park in Wyoming is
a brilliant display of blue-green algae.
Green chromoproteins called biliproteins are found in
many insects, such
as grasshoppers, and also in the eggshells of many birds. The biliproteins are
derived from the bile pigment biliverdin, which in turn is formed from
porphyrin; biliverdin contains four pyrrole rings and three of the four methine
groups of porphyrin. Large amounts of biliproteins have been found in red algae and blue-green algae;
the red protein is called phycoerythrin, the blue one phycocyanobilin.
When a protein solution is
mixed with a solution of a nucleic acid,
the phosphoric
acid component of the nucleic acid combines with
the positively charged ammonium groups (―NH3+) of the
protein to form a protein–nucleic acid complex. The nucleus of
a cell contains
predominantly deoxyribonucleic acid (DNA) and the cytoplasm predominantly
ribonucleic acid (RNA);
both parts of the cell also contain protein. Protein–nucleic acid complexes,
therefore, form in living cells.
The only nucleoproteins for which some evidence for
specificity exists are nucleoprotamines, nucleohistones, and some RNA and
DNA viruses. The
nucleoprotamines are the form in which protamines occur in the sperm cells of
fish; the histones of the thymus and of pea seedlings and other
plant material apparently occur predominantly as nucleohistones. Both
nucleoprotamines and nucleohistones contain only DNA.
Tobacco
mosaic virusSchematic structure of the tobacco mosaic virus. The
cutaway section shows the helical ribonucleic acid associated with protein
molecules in a ratio of three nucleotides per protein molecule.
Some of the simplest viruses consist of a specific RNA,
which is coated by protein. One of the best known RNA viruses, tobacco
mosaic virus (TMV),
has the shape of a rod. RNA comprises only
5.1 percent of the mass of the virus. The complete sequence of the virus
protein, which consists of about 2,130 identical peptide chains, each
containing 158 amino acids, has been determined. The protein is arranged in a
spiral around the RNA core.
DNA has been found in most bacterial viruses (bacteriophages) and
in some animal viruses. As in TMV, the core of DNA is surrounded by protein.
Phage protein is a mixture of enzymes and therefore cannot be considered as the
protein portion of only one nucleoprotein.
Respiratory proteins
Hemoglobin is the oxygen carrier in all
vertebrates and some invertebrates. In oxyhemoglobin (HbO2), which
is bright red, the ferrous ion (Fe2+)
is bound to the four nitrogen atoms
of porphyrin; the other two substituents are an oxygen molecule and
the histidine of
globin, the protein component of hemoglobin. Deoxyhemoglobin (deoxy-Hb), as its
name implies, is oxyhemoglobin minus oxygen (i.e., reduced hemoglobin); it is
purple in color. Oxidation of the ferrous ion of hemoglobin yields a
ferric compound,
methemoglobin, sometimes called hemiglobin or ferrihemoglobin. The oxygen of
oxyhemoglobin can be displaced by carbon monoxide,
for which hemoglobin has a much greater affinity,
preventing oxygen from reaching the body tissues.
The hemoglobins of all mammals, birds, and many
other vertebrates are
tetramers of two α- and two β-chains. The molecular weight of
the tetramer is 64,500; the molecular weight of the α- and β-chains is
approximately 16,100 each, and the four subunits are linked to each other by
noncovalent interactions. If hemin (the ferric porphyrin component) is removed
from globin (the protein component), two molecules of globin, each consisting
of one α- and one β-chain, are obtained; the molecular weight of globin is
32,200. In contrast to hemoglobin, globin is an unstable protein that is easily
denatured. If native globin is incubated with a solution of hemin at pH values
of 8 to 9, native hemoglobin is reconstituted. Myoglobin, the red pigment of
mammalian muscles, is a monomer with a
molecular weight of 16,000.
The mammalian hemoglobins differ from each other in
their amino acid composition and
therefore in their secondary and tertiary structure. Rat and horse hemoglobins
crystallize very easily, but those of humans, cattle, and sheep, because they
are more soluble, are difficult to crystallize. The shape of hemoglobin
crystals varies in different species; moreover, decomposition and denaturation occur
at different rates in different species. It was also found that the blood of human newborns
contains two different hemoglobins: about 20 percent of their hemoglobin is an
adult hemoglobin (hemoglobin A) and 80 percent is a fetal hemoglobin
(hemoglobin F). Hemoglobin F persists in the
infant for the first seven months of life. The same hemoglobin F has also been
found in the blood of patients suffering from thalassemia, an anemia with a high
incidence in regions surrounding the Mediterranean Sea.
Hemoglobin F contains, as does hemoglobin A, two α-chains; the two β-chains,
however, have been replaced by two quite different γ-chains. When the technique
of electrophoresis was
first applied to the hemoglobin of blacks suffering from sickle cell anemia in
1949, a new hemoglobin (hemoglobin S) was discovered. More than 200 different
human hemoglobins have been discovered since. They differ from normal
hemoglobin A in the amino acid composition of either the α- or the β-chain.
The hemoglobins of some of the lowest fishes are monomers
containing one iron atom per
molecule. Hemoglobin-like respiratory proteins have been found in some invertebrates. The
red hemoglobin of insects, mollusks, and protozoans is called erythrocruorin.
It differs from vertebrate hemoglobin
by its high molecular weight.
Although green plants contain no hemoglobin, a red protein,
called leghemoglobin, has been discovered in the root nodules of leguminous
plants. It seems to be produced by the nitrogen-fixing
bacteria of the root nodules and may be involved in the reduction of
atmospheric nitrogen to ammonia and amino acids.
Other respiratory proteins
A green respiratory protein, chlorocruorin, has been found
in the blood of marine worms in the genera Serpula and Spirographis.
It has the same high molecular weight as erythrocruorin but differs from
hemoglobin in its prosthetic group. A red metalloprotein, hemerythrin, acts as
a respiratory protein in marine worms of the phylum Sipuncula. The molecule
consists of eight subunits with a molecular weight of 13,500 each. Hemerythrin
contains no porphyrins and therefore is not a heme protein.
A metalloprotein containing copper is the respiratory
protein of crustaceans (shrimps, crabs, etc.) and of some gastropods (snails).
The protein, called hemocyanin,
is pale yellow when not combined with oxygen, and blue when combined with
oxygen. The molecular weights of hemocyanins vary from 300,000 to 9,000,000.
Each animal investigated thus far apparently has a species-specific hemocyanin.
Some hormones that are products of endocrine glands
are proteins or peptides, others are steroids. (The
origin of hormones, their physiological role, and their mode of action are
dealt with in the article hormone.) None of the
hormones has any enzymatic activity. Each has a target organ in which
it elicits some biological action—e.g., secretion of gastric or pancreatic
juice, production of milk, production of steroid hormones.
The mechanism by which the hormones exert their effects is not fully
understood. Cyclic adenosine monophosphate is involved in the transmittance of
the hormonal stimulus to the cells whose activity is specifically increased by
the hormone.
Hormones of the thyroid gland
Thyroglobulin, the active groups of which are two molecules
of the iodine-containing compound thyroxine, has a molecular weight of
670,000. Thyroglobulin also contains thyroxine with two and three iodine atoms
instead of four and tyrosine with one and two iodine atoms. Injection of the
hormone causes an increase in metabolism; lack of it
results in a slowdown.
Another hormone, calcitonin,
which lowers the calcium level of the blood, occurs
in the thyroid
gland. The amino
acid sequences of calcitonin from pig, beef, and salmon differ
from human calcitonin
in some amino acids. All of them, however, have the half-cystines (C) and the
prolinamide (P) in the same position.
Parathyroid hormone (parathormone),
produced in small glands that are embedded in or
lie behind the thyroid gland, is essential for maintaining the calcium level of
the blood. A decrease in its production results in hypocalcemia (a reduction of
calcium levels in the bloodstream below the normal range). Bovine parathormone
has a molecular weight of 8,500; it contains no cystine or cysteine and is
rich in aspartic
acid, glutamic
acid, or their amides.
Hormones of the pancreas
Although the amino acid structure of insulin has been
known since 1949, repeated attempts to synthesize it gave very poor yields
because of the failure of the two peptide chains to
combine forming the correct disulfide bridge. The ease of the biosynthesis of
insulin is explained by the discovery in the pancreas of
proinsulin, from which insulin is formed. The single peptide chain of
proinsulin loses a peptide consisting of 33 amino acids and called the
connecting peptide, or C peptide, during its conversion to insulin. The
disulfide bridges of proinsulin connect the A and B chains.
In aqueous solutions, insulin exists predominantly as a
complex of six subunits, each of which contains an A and a B chain. The
insulins of several species have been isolated and analyzed; their amino acid
sequences have been found to differ somewhat, but all apparently contain the
same disulfide bridges between the two chains.
Although the injection of insulin lowers the blood sugar,
administration of glucagon,
another pancreas hormone, raises the blood sugar level. Glucagon consists of a
straight peptide chain of 29 amino acids. It has been synthesized; the synthetic product
has the full biological activity of natural glucagon. The structure of glucagon
is free of cystine and isoleucine.
The pituitary gland has an anterior lobe, a posterior lobe,
and an intermediate portion; they differ in cellular structure and in the
structure and action of the hormones they form. The posterior lobe produces two
similar hormones, oxytocin and vasopressin. The former causes contraction of
the pregnant uterus; the latter raises the blood pressure. Both are
octapeptides formed by a ring of five amino acids (the two cystine halves count
as one amino acid) and a side chain of three amino acids. The two cystine
halves are linked to each other by a disulfide bond, and the C terminal amino
acid is glycinamide. The structure has been established and confirmed. Human
vasopressin differs from oxytocin in that isoleucine is replaced by phenylalanine and leucine by arginine.
The intermediate part of the pituitary gland produces
the melanocyte-stimulating
hormone (MSH), which causes expansion of the pigmented melanophores
(cells) in the skin of frogs and other batrachians. Two hormones, called α-MSH
and β-MSH, have been prepared from hog pituitary glands. The first, α-MSH,
consists of 13 amino acids; its N terminal serine
is acetylated (i.e., the acetyl group, CH3CO, of acetic acid is
attached), and its C terminal valine residue is
present as valinamide. The second, β-MSH, contains in its 18 amino acids many
of those occurring in α-MSH.
The anterior pituitary lobe produces several protein
hormones—a thyroid-stimulating hormone (thyrotropin),
molecular weight 28,000; a lactogenic hormone, molecular weight 22,500; a growth hormone,
molecular weight 21,500; a luteinizing
hormone, molecular weight 30,000; and a follicle-stimulating
hormone, molecular weight 29,000. The thyroid-stimulating hormone consists
of α and β subunits with a composition similar
to the subunits of luteinizing hormone. When separated, neither of the two
subunits has hormonal activity; when combined, however, they regain about 50
percent of the original activity. The lactogenic hormone (prolactin) from sheep
pituitary glands contains 190 amino acids. Their sequence has been elucidated;
a similar peptide chain of 188 amino acids that has been synthesized not only
has 10 percent of the biological activity of the natural hormone but also some
activity of the growth hormone. The amino acid sequence of the growth hormone
(somatotropic hormone) is also known; it seems to stimulate the synthesis
of RNA and in
this way to accelerate growth. The luteinizing hormone, a mucoprotein
containing about 12 percent carbohydrate,
consists of two subunits, each with a molecular weight of approximately 15,000;
when separated, the subunits recombine spontaneously. The urine of pregnant
women contains chorionic gonadotropin, the
presence of which makes possible early diagnosis of pregnancy. The amino
acid sequence is known. The sequence of 160 of its 190 amino acids is identical
with those of the growth hormone; 100 of these also occur in the same sequence
as in lactogenic hormone. The different pituitary hormones and the chorionic
gonadotropin thus may have been derived from a common substance that,
during evolution,
underwent differentiation.
Peptides with
hormonelike activity
Small peptides have been discovered that, like hormones, act
on certain target organs. One peptide, angiotensin (angiotonin
or hypertensin), is formed in the blood from angiotensinogen by the action
of renin, an enzyme of the kidney. It is an
octapeptide and increases blood pressure.
Similar peptides include bradykinin, which stimulates smooth muscles; gastrin, which stimulates
secretion of hydrochloric
acid and pepsin in
the stomach; secretin,
which stimulates the flow of pancreatic juice; and kallikrein, the activity of
which is similar to bradykinin.
Immunoglobulins and
antibodies
Antibodies,
proteins that combat foreign substances in the body, are associated with
the globulin fraction
of the immune serum.
As stated previously, when the serum globulins are separated into α-, β-, and
γ- fractions, antibodies are associated with the γ-globulins. Antibodies can be
purified by precipitation with the antigen (i.e., the
foreign substance) that caused their formation, followed by separation of the
antigen-antibody complex. Antibodies prepared in this way consist of a mixture
of many similar antibody molecules, which differ
in molecular
weight, amino
acid composition,
and other properties. The same differences are found in the γ-globulins of
normal blood serums. The γ-globulin of
normal blood serum is thought to consist of a mixture of hundreds of different
γ-globulins, each of which occurs in amounts too small for isolation. Because
the physical and chemical properties of normal γ-globulins are the same as those
of antibodies, the γ-globulins are frequently called immunoglobulins. They may
be considered to be antibodies against unknown antigens. If solutions of
γ-globulin are resolved by gel filtration through
dextran, the first fraction has a molecular weight of 900,000. This fraction is
called IgM or γM; Ig is an abbreviation for immunoglobulin and M for
macroglobulin. The next two fractions are IgA (γA) and IgG (γG), with molecular
weights of about 320,000 and 150,000 respectively. Two other immunoglobulins,
known as IgD and IgE, have also been detected in much smaller amounts in
some immune sera.
The bulk of the immunoglobulins is found in the IgG
fraction, which also contains most of the antibodies. The IgM molecules are
apparently pentamers—aggregates of five of the IgG molecules. Electron
microscopy shows their five subunits to be linked to each other by
disulfide bonds in the form of a pentagon. The IgA molecules are found
principally in milk and
in secretions of the intestinal mucosa. Some of them contain, in addition to a
dimer of IgG, a “secretory piece” that enables the passage of IgA molecules
between tissue and fluid; the structure of the secretory piece is not yet known. The IgM
and IgA immunoglobulins and antibodies contain 10 to 15 percent carbohydrate; the
carbohydrate content of the IgG molecules is 2 to 3 percent.
IgG
immunoglobulinDiagram of an IgG immunoglobulin.
IgG molecules treated with the enzyme papain split into
three fragments of almost identical molecular weight of 50,000. Two of these,
called Fab fragments, are identical; the third is abbreviated Fc. Reduction to
sulfhydryl groups of some of the disulfide bonds of IgG results in the
formation of two heavy, or H, chains (molecular weight 55,000) and
two light, or L, chains (molecular weight 22,000). They are linked
by disulfide bonds in the order L―H―H―L.
Each H chain contains four intrachain disulfide bonds, and
each L chain contains two.
Antibody preparations of the IgG type, even after removal of
IgM and IgA antibodies, are heterogeneous.
The H and L chains consist of a large number
of different L chains and a variety of H chains.
Pure IgG, IgM, and IgA immunoglobulins, however, occur in the blood serum of
patients suffering from myelomas, which are malignant tumors of the bone marrow. The
tumors produce either an IgG, an IgM, or an IgA protein, but rarely more than
one class. A protein called the Bence-Jones
protein, which is found in the urine of patients suffering from myeloma
tumors, is identical with the L chains of the myeloma protein.
Each patient has a different Bence-Jones protein; no two of the more than 100
Bence-Jones proteins that have been analyzed thus far are identical. It is
thought that one lymphoid cell among
hundreds of thousands becomes malignant and
multiplies rapidly, forming the mass of a myeloma tumor that produces one
γ-globulin.
Analyses of the Bence-Jones proteins have revealed that
the L chains of humans and other mammals are of two
quite different types, kappa (κ) and lambda (λ). Both consist of approximately
220 amino acids. The N–terminal halves of κ- and λ-chains are variable,
differing in each Bence-Jones protein. The C–terminal halves of these
same L chains have a constant amino acid sequence of either
the κ- or the λ-type. The fact that one half of a peptide chain is
variable and the other half invariant is contradictory to the view that the
amino acid sequence of each peptide chain is determined by one gene. Evidently, two genes,
one of them variable, the other invariant, fuse to form the gene for the single
peptide chain of the L chains. Whereas the normal human L chains
are always mixtures of the κ- and λ-types, the H chains of
IgG, IgM, and IgA are different. They have been designated as
gamma (γ), mu (μ), and alpha (α) chains, respectively. The N-terminal quarter
of the H chains has a variable amino acid sequence; the
C-terminal three-quarters of the H chains have a constant
amino acid sequence.
Some of the amino acid sequences in the L and H chains
are transmitted from generation to generation. As a result, the constant
portion of the human L chains of the κ-type has in position
191 either valine or leucine. They correspond
to two alleles (character-determining
portions) of a gene; the two types are called allotypes. The valine-containing
genetic type has been designated as InV(a+), the leucine-containing
type as InV(b+). Many more allotypes, called Gm allotypes, have been
found in the gamma chains of the human IgG immunoglobulins; more than 20 Gm
allotypes are known. Certain combinations of Gm types occur. For example, the
combination of Gm types 5, 6, and 11 has been found in Caucasians and African Americans but
not in Chinese; the combination of 1, 2, and 17 has not been found in African
Americans; and the combination of 1, 4, and 17 has not been found in
Caucasians. Allotypes have also been discovered to occur in a number of other
animals, including rabbits and mice.
It is understandable from the occurrence of a large number
of allotypes that antibodies, even if produced in response to a single antigen,
are mixtures of different allotypes. The existence of several classes of
antibodies, of different allotypes, and of adaptation of
the variable portions of antibodies to different regions of an antigen molecule results in
a multiplicity of antibody molecules even if only a single antigen is
administered. For this reason it has not yet been possible to unravel the amino
acid sequence in the variable portion of antibody molecules. Much of the amino
acid sequence in the constant regions of the L and H chains
of humans and rabbit immunoglobulins, however, has been resolved.
Felix
Haurowitz The
Editors of Encyclopaedia Britannica
Practically all of the numerous and complex biochemical
reactions that take place in animals, plants, and microorganisms
are regulated by enzymes. These catalytic proteins are efficient and
specific—that is, they accelerate the rate of one kind of chemical reaction of
one type of compound,
and they do so in a far more efficient manner than human-made catalysts. They are
controlled by activators and inhibitors that initiate or block reactions.
All cells contain
enzymes, which usually vary in number and composition,
depending on the cell type;
an average mammalian cell, for example, is approximately one one-billionth (10−9)
the size of a drop of water and
generally contains about 3,000 enzymes.
The existence of enzymes was established in the middle of
the 19th century by scientists studying the process of fermentation. The
discovery of the role of enzymes as catalysts followed
rapidly. Developments before 1850 included (in 1833) the separation from malt
of the enzyme amylase, which converts
starch into sugar, and (in 1836) the isolation from the stomach wall of animals
of a component of gastric juice that could partially digest food in a test tube, the
enzyme pepsin.
Enzymes were known for many years as ferments, a
term derived from the Latin word for yeast. In 1878 the
name enzyme, from the Greek words meaning “in yeast,” was
introduced; since the late 19th century it has been employed universally.
Role of enzymes in metabolism
Some enzymes help to break down large nutrient molecules,
such as proteins, fats,
and carbohydrates,
into smaller molecules. This process occurs during the digestion of
foodstuffs in the stomach and intestines of animals. Other enzymes guide the
smaller, broken-down molecules through the intestinal wall into the
bloodstream. Still other enzymes promote the formation of large, complex
molecules from the small, simple ones to produce cellular constituents.
Enzymes are also responsible for numerous other functions, which include the
storage and release of energy, the course of reproduction, the processes of
respiration, and vision. They are indispensable to life.
Each enzyme is able to promote only one type of chemical reaction.
The compounds on
which the enzyme acts are called substrates.
Enzymes operate in tightly organized metabolic systems called pathways. A
seemingly simple biological phenomenon—the contraction of a muscle, for example, or
the transmission of a nerve impulse—actually involves a large number of
chemical steps in which one or more chemical compounds (substrates)
are converted to substances called products; the product of one step in a
metabolic pathway serves as the substrate for the succeeding step in the
pathway.
The role of enzymes in metabolic pathways can be illustrated
diagrammatically. The chemical compound represented
by A (see diagram below) is converted to
product E in a series of enzyme-catalyzed steps, in which
intermediate compounds represented by B, C, and D are
formed in succession. They act as substrates for enzymes represented by 2, 3,
and 4. Compound A may also be converted by another series of
steps, some of which are the same as those in the pathway for the formation
of E, to products represented by G and H.
The letters represent chemical compounds; numbers represent
enzymes that catalyze individual reactions. The relative heights represent the
thermodynamic energy of the compounds (e.g., compound A is
more energy-rich than B, B more energy-rich
than C). Compounds A, B, etc., change very
slowly in the absence of a catalyst but
do so rapidly in the presence of catalysts 1,
2, 3, etc.
The regulatory role of enzymes in metabolic pathways can be
clarified by using a simple analogy: that between the compounds, represented by
letters in the diagram, and a series of connected water reservoirs on a
slope. Similarly, the enzymes represented by the numbers are analogous to
the valves of the reservoir system. The valves control the flow of water in the
reservoir; that is, if only valves 1, 2, 3, and 4 are open, the water in A flows
only to E, but, if valves 1, 2, 5, and 6 are open, the water
in A flows to G. In a similar manner, if enzymes
1, 2, 3, and 4 in the metabolic pathway are active, product E is
formed, and, if enzymes 1, 2, 5, and 6 are active, product G is
formed. The activity or lack of activity of the enzymes in the pathway
therefore determines the fate of compound A; i.e., it either
remains unchanged or is converted to
one or more products. In addition, if products are formed, the activity of
enzymes 3 and 4 relative to that of enzymes 5 and 6 determines the quantity of
product E formed compared with product G.
Both the flow of water and the activity of enzymes obey the
laws of thermodynamics; hence, water in reservoir F cannot
flow freely to H by opening valve 7, because water cannot flow
uphill. If, however, valves 1, 2, 5, and 7 are open, water flows from F to H,
because the energy conserved during the downhill flow of water through valves
1, 2, and 5 is sufficient to allow it to force the water up through valve 7. In
a similar way, enzymes in the metabolic pathway cannot convert compound F directly
to H unless energy is available; enzymes are able to utilize
energy from energy-conserving reactions in order to catalyze reactions that
require energy. During the enzyme-catalyzed oxidation of carbohydrates to carbon dioxide and
water, energy is conserved in the form of an energy-rich compound, adenosine
triphosphate (ATP). The energy in ATP is utilized during an
energy-consuming process such as the enzyme-catalyzed contraction of muscle.
Because the needs of cells and organisms vary, not only the
activity but also the synthesis of enzymes must be regulated; e.g., the enzymes
responsible for muscular activity in a leg muscle must be activated and inhibited at
appropriate times. Some cells do not need certain enzymes; a liver cell, for example,
does not need a muscle enzyme. A bacterium does not
need enzymes to metabolize substances that are not present in its growth medium. Some
enzymes, therefore, are not formed in certain cells, others are synthesized
only when required, and still others are found in all cells. The formation and
activity of enzymes are regulated not only by genetic mechanisms but also by
organic secretions (hormones)
from endocrine glands and by nerve impulses. Small molecules also play an
important role (see below Enzyme
flexibility and allosteric control).
If an enzyme is defective in some respect, disease may
occur. The enzymes represented by the numbers 1 to 4 in the diagram must
function during the conversion of
the starting substance A to the product E. If one
step is blocked because an enzyme is unable to function, product E may
not be formed; if E is necessary for some vital function,
disease results. Many inherited diseases and conditions of humans result from a
deficiency of one enzyme. Some of these are listed in the table. Albinism, for example,
results from an inherited lack of ability to synthesize the enzyme tyrosinase,
which catalyzes one step in the pathway by which the pigment for hair and eye
color is formed.
|
Enzymes
identified with hereditary diseases |
|
|
disease
name |
defective
enzyme |
|
albinism |
tyrosinase |
|
phenylketonuria |
phenylalanine
hydroxylase |
|
fructosuria |
fructokinase |
|
methemoglobinemia |
methemoglobin
reductase |
|
galactosemia |
galactose-1-phosphate
uridyl transferase |
Other functions
Enzymes play an increasingly important role in medicine. The enzyme thrombin is
used to promote the healing of wounds. Other enzymes are
used to diagnose certain kinds of disease, to cause the remission of some forms
of leukemia—a
disease of the blood-forming organs—and to counteract unfavorable reactions in
people who are allergic to penicillin. The
enzyme lysozyme,
which destroys cell walls,
is used to kill bacteria.
Enzymes have also been investigated for their potential to prevent tooth decay and to
serve as anticoagulants in
the treatment of thrombosis,
a disease characterized by the formation of a clot, or plug, in a blood vessel.
Enzymes may eventually be used to control enzyme deficiencies and abnormalities
resulting from diseases.
It might also be noted in passing that enzymes are used in
industrial processes involving the preparation of certain chemical compounds and
the tanning of leather. They also are valuable in analytical procedures
involving the detection of very small quantities of specific substances.
Enzymes are necessary in various food-related industries, including cheese making, the
brewing of beer, the
aging of wine, and the
baking of bread.
Enzymes also may be used to clean clothes. For some industrial uses of
enzymes, see baking.
General properties
Classification and nomenclature
The first enzyme name, proposed in 1833, was diastase.
Sixty-five years later, French microbiologist and chemist Émile Duclaux
suggested that all enzymes be named by adding -ase to a root
indicative of the nature of the substrate of the enzyme. Although enzymes are
no longer named in such a simple manner, with the exception of a few—e.g.,
pepsin, trypsin, chymotrypsin, papain—most enzyme names do end in -ase.
Any systematic classification
of enzymes should be based on a common property or quality that varies
sufficiently to be useful as a distinguishing feature. In this regard, three
properties of enzymes could serve as a basis for enzyme classification—the
exact chemical nature of the enzyme, the chemical nature of the substrate, and
the nature of the reaction catalyzed. In addition, although, as indicated
above, early attempts at enzyme classification were based on the nature of
broad groups of substrates (e.g., enzymes called carbohydrases act on
carbohydrates), close functional similarities among enzymes in different groups
were often obscured. By general agreement, enzymes now are classified according
to their substrates and the nature of the reaction they catalyze.
In an attempt to devise a rational system of enzyme nomenclature,
two names are given to an enzyme. One, known as the systematic name, is based
on logical principles but is often long and awkward; the other, “trivial” name
is short and generally used but not usually exact or systematic. In the scheme
of systematic nomenclature, six main groups of enzymatic reactions are
recognized; each catalyzes one reaction type and is subdivided on the basis of
detailed definitions of the reaction catalyzed and of the substrate involved in
the reaction. Enzymes that catalyze reactions in which hydrogen is
transferred belong to the group known as oxidoreductases;
those that catalyze the introduction of the elements of water at a specific
site in a molecule are
called hydrolases.
The other four groups of reactions are the transferases—which
catalyze reactions in which substances other than hydrogen are
transferred—the lyases,
the isomerases,
and the ligases.
Oxidoreductases and transferases account for about 50 percent of the
approximately 1,000 enzymes recognized thus far. The table lists a few
enzymes, their trivial names,
their systematic names, and their biological roles.
|
Classification
of some enzymes |
||||
|
systematic
name* |
trivial
name |
reaction
catalyzed |
biological
role |
|
|
code
number** |
name*** |
|||
|
1.1.1.1 |
alcohol:
NAD oxidoreductase |
alcohol
dehydrogenase |
alcohol
+ NAD → acetaldehyde NADH |
alcoholic
fermentation |
|
1.1.1.27 |
L-lactate:
NAD oxidoreductase |
lactic
dehydrogenase |
lactate
+ NAD → pyruvate + NADH |
carbohydrate
metabolism |
|
2.7.1.40 |
ATP:
pyruvate phosphotransferase |
pyruvate
kinase |
pyruvic
acid + ATP → phosphoenolpyruvic acid + ADP |
carbohydrate
metabolism |
|
3.1.1.7 |
acetylcholine:
acetylhydrolase |
acetylcholinesterase |
acetylcholine
+ H2O → acetate + choline |
nerve-impulse
conduction |
|
*Based
on recommendations (1964) of the International Union of Biochemistry. |
||||
Chemical nature
Little was known about the chemical nature of enzymes until
the beginning of the 20th century, although scientists were almost convinced
that they were proteins. In 1926 the enzyme urease was the first
to be crystallized and clearly identified as a protein. Within the next few
years the digestive enzymes pepsin, trypsin, and chymotrypsin were shown to be
proteins. Since that time hundreds of enzymes, all of them proteins, have been prepared
and characterized by chemical methods. Much of the knowledge of protein chemistry has, in
fact, resulted from studies involving enzymes and from attempts to understand
their nature and mode of action.
Although some enzymes consist of a single chain of the amino acids (i.e.,
simple organic molecules containing nitrogen), most enzymes are composed of
more than one chain. Each chain is called a subunit. Many enzymes have two,
four, or six subunits, and some consist of as many as 12 to 60 subunits. In
many cases the subunits have identical structures; in others, however, several
different types of subunit chains are involved.
With the exception of proteins that act as structural
elements, most of the proteins in physiologically active tissues such as kidney and liver are enzymes.
Regardless of the exact amount of enzymatic protein in an organism, it is clear
that hundreds of different enzymes must be present in each tissue to account
for the myriad reactions
composing metabolism.
B-vitamin
coenzymes in metabolismFunctions of B-vitamin coenzymes in metabolism.
Although some enzymes consist only of protein, many are
complex proteins; i.e., they have a protein component and a so-called cofactor. A
complete enzyme is
called a holoenzyme; if the cofactor is removed, the protein, no longer
enzymatically active, is called the apoenzyme. A cofactor may be a metal—such
as iron, copper, or magnesium—a moderately
sized organic molecule called
a prosthetic group, or a special type of substrate molecule known as a coenzyme. The cofactor
may aid in the catalytic function of an enzyme, as do metals and prosthetic
groups, or take part in the enzymatic reaction, as do coenzymes.
A coenzyme serves as a type of substrate in certain
enzymatic reactions and thus reacts in the exact proportions (i.e.,
stoichiometrically) required for reaction, rather than in catalytic quantities.
A coenzyme may, for example, assume the role of a hydrogen acceptor, as does
nicotinamide adenine dinucleotide
(NAD), which accepts hydrogen from the substrate, or a chemical-group donor, as
does adenosine
triphosphate (ATP), which donates phosphoric acid to
the substrate. After ATP has donated a phosphoric acid molecule to the
substrate, the phosphoric acid can be reacquired in a second stoichiometric
reaction catalyzed by a second enzyme. The catalytic nature of a coenzyme is
apparent only when it couples the activities of two enzymes in this way.
Coenzymes thus are the links, or shuttles, in metabolic pathways that enable
substances—e.g., hydrogen, phosphoric acid—to be exchanged.
The nature of enzyme-catalyzed reactions
The nature of catalysis
In a chemical reaction—for example, one in which
substance A is converted into product B—a point
of equilibrium eventually
is reached at which no further chemical change occurs; i.e., the rate of
conversion of A to B equals the rate of
conversion of B to A. The so-called
thermodynamic-equilibrium constant expresses this chemical
equilibrium. A catalyst may
be defined as a substance that accelerates a chemical reaction but
is not consumed in the process. The amount of catalyst has no relationship to
the quantity of substance altered; very small amounts of enzymes are very
efficient catalysts.
Because the presence of an enzyme accelerates the rate of conversion of a compound to
a product, it accelerates the approach to equilibrium; it does not, however,
influence the equilibrium point attained.
The molecules in the watery medium of the cell are in
constant thermal motion but, because they are more or less stable compounds, they
would react only occasionally to form products in the absence of enzymes. There
exists an energy barrier to the reaction of a molecule. The energy required to
overcome the barrier to reaction is called the energy of activation. A reaction
proceeds to equilibrium only if the molecules have sufficient energy of
activation to form an activated complex, from which products can be derived.
Enzymes greatly increase the chances for reactions by their ability to make
large numbers of specific molecules more reactive (i.e., unstable) by forming
intermediate compounds with them. The unstable intermediates quickly break down
to form stable products, and the enzymes, unchanged by the reaction, are able
to catalyze the formation of additional products.
The role of the active site
That the compound on
which an enzyme acts
(substrate) must combine in some way with it before catalysis can proceed is an
old idea, now supported by much experimental evidence. The combination of
substrate molecules with enzymes involves collisions between the two. Enzymes
are large molecules, the molecular weights of which (based on the weight of a
hydrogen atom as
1) range from several thousand to several million. The substrates on which
enzymes act usually have molecular weights of several hundred. Because of the
difference in size between the two, only a fraction of the enzyme is in contact
with the substrate; the region of contact is called the active site. Usually,
each subunit of an enzyme has one active site capable of binding substrate.
Enzymes
and their active sitesThe role of the active site in the lock-and-key
fit of a substrate (the key) to an enzyme (the lock).
The characteristics of
an enzyme derive from the sequence of amino acids, which determine the shape of
the enzyme (i.e., the structure of the active site) and hence the specificity
of the enzyme. The forces that attract the substrate to the surface of an
enzyme may be of a physical or a chemical nature. Electrostatic bonds may occur
between oppositely charged groups—the circles containing plus and minus signs
on the enzyme are attracted to their opposites in the substrate molecule. Such
electrostatic bonds can occur with groups that are completely positively or
negatively charged (i.e., ionic groups) or with groups that are partially
charged (i.e., dipoles). The attractive forces between substrate and enzyme may
also involve so-called hydrophobic bonds, in which the oily, or hydrocarbon, portions
of the enzyme (represented by H-labelled circles) and the substrate are forced
together in the same way as oil droplets tend to coalesce in water.
Modifications in the structure of the amino acids at or near
the active site usually affect the enzyme’s activity, because these amino acids
are intimately involved in the fit and attraction of the substrate to the
enzyme surface. The characteristics of the amino acids near the active site
determine whether or not a substrate molecule will fit into the site. A
molecule that is too bulky in the wrong places cannot fit into the active site
and thus cannot react with the enzyme. In a similar manner, a molecule lacking
essential attractive forces or the appropriately charged regions might not be
bound to the enzyme. On the other hand, a molecule with a bulky group at a
position such that it does not interfere with the binding of the molecule to
the enzyme or with the function of the active site is able to serve as a
substrate for the enzyme. The idea of a fit between substrate and enzyme,
called the “key–lock” hypothesis,
was proposed by German chemist Emil Fischer in
1899 and explains one of the most important features of enzymes, their
specificity. In most of the enzymes studied thus far, a cleft, or
indentation, into which the substrate fits is found at the active site.
The specificity of enzymes
Since the substrate must fit into the active site of
the enzyme before
catalysis can occur, only properly designed molecules can serve as substrates
for a specific enzyme; in many cases, an enzyme will react with only one
naturally occurring molecule.
Two oxidoreductase enzymes will serve to illustrate the principle of enzyme
specificity. One (alcohol dehydrogenase) acts on alcohol, the other
(lactic dehydrogenase) on lactic acid; the
activities of the two, even though both are oxidoreductase enzymes, are not
interchangeable—i.e., alcohol dehydrogenase will not catalyze a reaction
involving lactic acid or vice versa, because the structure of each substrate
differs sufficiently to prevent its fitting into the active site of the alternative enzyme.
Enzyme specificity is essential because it keeps separate the many pathways,
involving hundreds of enzymes, that function during metabolism.
Not all enzymes are highly specific. Digestive enzymes such
as pepsin and chymotrypsin,
for example, are able to act on almost any protein, as they must if they are to
act upon the varied types of proteins consumed as food. On the other hand, thrombin, which reacts
only with the protein fibrinogen, is part of a very delicate blood-clotting
mechanism and thus must act only on one compound in
order to maintain the proper functioning of the system.
When enzymes were first studied, it was thought that most of
them were “absolutely specific”—that they would react with only one compound.
In most cases, however, a molecule other than the natural substrate can be
synthesized in the laboratory; it is enough like the natural substrate to react
with the enzyme. Use of these synthetic substrates
has been valuable in understanding enzymatic action. It must be remembered,
however, that, in the living cell, many enzymes
are absolutely specific for the compounds found
there.
All enzymes isolated thus far are specific for the type
of chemical
reaction they catalyze—i.e., oxidoreductases do not catalyze hydrolase
reactions, and hydrolases do not catalyze reactions involving oxidation and
reduction. An enzyme therefore catalyzes a specific chemical reaction but may
be able to do so on several similar compounds.
The mechanism of enzymatic action
The
actions of enzymesMechanisms of enzymatic action.
An enzyme attracts
substrates to its active site, catalyzes the chemical reaction by
which products are formed, and then allows the products to dissociate (separate
from the enzyme surface). The combination formed by an enzyme and its
substrates is called the enzyme–substrate complex. When two substrates and one
enzyme are involved, the complex is called a ternary complex; one substrate and
one enzyme are called a binary complex.
The substrates are attracted to the active site by electrostatic and
hydrophobic forces, which are called noncovalent bonds because they are
physical attractions and not chemical bonds.
As an example, assume two substrates (S1 and S2)
bind to the active site of the enzyme during step 1 and react to form products
(P1 and P2) during step 2. The
products dissociate from the enzyme surface in step 3, releasing the enzyme.
The enzyme, unchanged by the reaction, is able to react with additional
substrate molecules in this manner many times per second to form products. The
step in which the actual chemical transformation occurs is of great interest,
and, although much is known about it, it is not yet fully understood. In
general there are two types of enzymatic mechanisms, one in which a so-called
covalent intermediate forms and one in which none forms.
In the mechanism by which a covalent intermediate—i.e., an
intermediate with a chemical bond between substrate and enzyme—forms, one
substrate, B―X, for example, reacts with the group N on
the enzyme surface to form an enzyme-B intermediate compound. The
intermediate compound then reacts with the second substrate, Y, to
form the products B―Y and X.
Many enzymes catalyze reactions by this type of
mechanism. Acetylcholinesterase is
used as a specific example in the sequence described below. The two substrates
(S1 and S2) for
acetylcholinesterase are acetylcholine (i.e., B―X) and water (Y).
After acetylcholine (B―X) binds to the enzyme surface, a chemical
bond forms between the acetyl moiety (B) of acetylcholine and the
group N (part of the amino acid serine)
on the enzyme surface. The result of the formation of this bond, called an
acyl–serine bond, is one product, choline (X), and the enzyme-B intermediate
compound (an acetyl–enzyme complex). The water molecule (Y)
then reacts with the acyl–serine bond to form the second product, acetic acid (B―Y),
which dissociates from the enzyme. Acetylcholinesterase is regenerated and is
again able to react with another molecule of acetylcholine. This kind of
reaction, involving the formation of an intermediate compound on the enzyme
surface, is generally called a double displacement
reaction.
Sucrose phosphorylase acts in a similar way. The substrate
for sucrose phosphorylase is sucrose, or glucosyl-fructose (B―X),
and the group N on the enzyme surface is a chemical group
called a carboxyl group (COOH). The enzyme-B intermediate, a
glucosyl–carboxyl compound, reacts with phosphate (Y) to form
glucosyl-phosphate (B―Y). The other product (X) is
fructose.
In double displacement reactions, the covalent intermediate
between enzyme and substrate apparently influences the reaction to proceed more
rapidly. Because the enzyme is unaltered at the end of the reaction, it
functions as a true catalyst, even
though it is temporarily altered during the enzymatic process.
Although many enzymes form a covalent intermediate, the
mechanism is not essential for catalysis. One substrate (Y) reacts
directly with the second substrate (X―B), in a so-called single
displacement reaction. The B moiety, which is transformed in
the chemical reaction, is involved in only one reaction and does not form a
bond with a group on the enzyme surface. The enzyme maltose phosphorylase, for
example, directly affects the bonds of the substrates (B―X and X),
which, in this case, are maltose (glucosylglucose) and phosphate, to form the
products, glucose (X)
and glucosylphosphate (B―Y).
Covalent intermediates between part of a substrate and an
enzyme occur in many enzymatic reactions, and various amino acids—serine,
cysteine, lysine,
and glutamic acid—are involved.
The rate of enzymatic reactions
The Michaelis-Menten
hypothesis
Diagram
of enzyme actionCurves representing enzyme action.
If the velocity of an enzymatic reaction is represented
graphically as a function of the substrate concentration (S), the curve
obtained in most cases is a hyperbola. The mathematical expression of this
curve, shown in the equation below, was developed in 1912–13 by German
biochemists Leonor Michaelis and Maud Leonora
Menten. In the equation, VM is the maximal
velocity of the reaction, and KM is called the
Michaelis constant,
The shape of the curve is a logical consequence of the
active-site concept; i.e., the curve flattens at the maximum velocity (VM),
which occurs when all the active sites of the enzyme are filled with substrate.
The fact that the velocity approaches a maximum at high substrate
concentrations provides support for the assumption that an intermediate
enzyme–substrate complex forms. At the point of half the maximum velocity, the
substrate concentration in moles per
litre (M) is equal to the Michaelis constant, which is a rough measure
of the affinity of
the substrate molecule for the surface of the enzyme. KM values
usually vary from about 10−8 to 10−2 M,
and VM from 105 to 109 molecules
of product formed per molecule of enzyme per second. The value for VM is
referred to as the turnover number when expressed as moles of product formed
per mole of enzyme per minute. The binding of molecules that inhibit or
activate the protein surface usually results in similar types.
Enzymes are more efficient than human-made catalysts operating
under the same conditions. Because many enzymes with different specificities
occur in a cell,
adequate space exists only for a few enzyme molecules catalyzing one specific
reaction. Each enzyme, therefore, must be very efficient. One molecule of the
enzyme catalase,
for example, can produce 1012 molecules of oxygen per second.
The catalytic groups at the active site of an enzyme act 106 to
109 times more effectively than do analogous groups
in a nonenzymatic reaction.
The reason for the great efficiency of
enzymes is not completely understood. It results in part from the precise
positioning of the substrates and the catalytic groups at the active site,
which serves to increase the probability of collision between the reacting
atoms. In addition, the environment at
the active site may be favorable for reaction—that is, acidic and basic groups
may act together more effectively there, or some strain may be induced in the
substrate molecules so that their bonds are broken more easily, or the
orientation of the reacting substrates may be optimal at the enzyme surface.
The theories that have been formulated to account for the high catalytic
efficiency of enzymes, although reasonable, still remain to be proved.
Inhibition of enzymes
Some molecules very similar to the substrate for an enzyme may be bound
to the active site but be unable to react. Such molecules cover the active site
and thus prevent the binding of the actual substrate to the site. This inhibition of
enzyme action is of a competitive nature, because the inhibitor molecule actually
competes with the substrate for the active site. The inhibitor sulfanilamide, for
example, is similar enough to a substrate (p-aminobenzoic acid) of an
enzyme involved in the metabolism of folic acid that
it binds to the enzyme but cannot react. It covers the active site and prevents
the binding of p-aminobenzoic acid. This enzyme is
essential in certain disease-causing bacteria but is not
essential to humans; large amounts of sulfanilamide therefore kill the
microorganism but do not harm humans. Inhibitors such as sulfanilamide are
called antimetabolites.
Sulfanilamide and similar compounds that
kill a pathogen without harming its host are widely used in chemotherapy.
Some inhibitors prevent, or block, enzymatic action by
reacting with groups at the active site. The nerve gas diisopropyl
fluorophosphate, for example, reacts with the serine at the active site of
acetylcholinesterase to form a covalent bond. The
nerve gas molecule involved in bond formation prevents the active site from
binding the substrate, acetylcholine,
thereby blocking catalysis and nerve action. Iodoacetic acid similarly blocks a
key enzyme in muscle action
by forming a bulky group on the amino acid cysteine,
which is found at the enzyme’s active site. This process is called
irreversible inhibition.
Some inhibitors modify amino acids other than those at the
active site, resulting in loss of enzymatic activity. The inhibitor causes
changes in the shape of the active site. Some amino acids other than those at
the active site, however, can be modified without affecting the structure of
the active site; in these cases, enzymatic action is not affected.
Such chemical changes parallel natural mutations. Inherited
diseases frequently result from a change in an amino acid at the active site of
an enzyme, thus making the enzyme defective. In some cases, an amino acid
change alters the shape of the active site to the extent that it can no longer
react; such diseases are usually fatal. In others, however, a partially
defective enzyme is formed, and an individual may be very sick but able to
live.
Effects of temperature
Enzymes function most efficiently within a physiological
temperature range. Since enzymes are protein molecules, they can be destroyed
by high temperatures. An example of such destruction, called protein denaturation, is the
curdling of milk when it is boiled. Increasing temperature has two effects on
an enzyme: first, the velocity of the reaction increases somewhat, because the
rate of chemical reactions tends to increase with temperature; and, second, the
enzyme is increasingly denatured. Increasing temperature thus increases the
metabolic rate only within a limited range. If the temperature becomes too
high, enzyme denaturation destroys life. Low temperatures also change the
shapes of enzymes. With enzymes that are cold-sensitive, the change causes loss
of activity. Both excessive cold and heat are therefore damaging to enzymes.
The degree of acidity or basicity of a solution,
which is expressed as pH,
also affects enzymes. As the acidity of a solution changes—i.e., the pH is
altered—a point of optimum acidity occurs, at which the enzyme acts most
efficiently. Although this pH optimum varies with temperature and is influenced
by other constituents of
the solution containing the enzyme, it is a characteristic property of enzymes.
Because enzymes are sensitive to changes in acidity, most living systems are
highly buffered; i.e., they have mechanisms that enable them to maintain a
constant acidity. This acidity level, or pH, is about 7 in most organisms. Some
bacteria function under moderately acidic or basic conditions; and the
digestive enzyme pepsin acts in the acid milieu of
the stomach.
Enzyme flexibility and allosteric control
The key–lock
hypothesis (see above The nature
of enzyme-catalyzed reactions) does not fully account for enzymatic action;
i.e., certain properties of enzymes cannot be accounted for by the simple
relationship between enzyme and substrate proposed by the key–lock hypothesis. A
theory called the induced-fit theory retains the key–lock idea of a fit of the
substrate at the active site but postulates in addition that the substrate must
do more than simply fit into the already preformed shape of an active site.
Rather, the theory states, the binding of the substrate to the enzyme must
cause a change in the shape of the enzyme that results in the proper alignment
of the catalytic groups on its surface. This concept has been
likened to the fit of a hand in a glove, the hand (substrate) inducing a change
in the shape of the glove (enzyme). Although some enzymes appear to function
according to the older key–lock hypothesis, most apparently function according
to the induced-fit theory.
Induced-fit
theoryVarious anomalous properties of enzymes are explained by the
induced-fit binding theory, wherein a substrate binds to an enzyme surface,
triggering allosteric effects.
Typically, the substrate approaches the enzyme surface and
induces a change in its shape that results in the correct alignment of the
catalytic groups. In the case of the digestive enzyme carboxypeptidase,
for example, the binding of the substrate causes a tyrosine molecule at the
active site to move by as much as 15 angstroms. The catalytic groups at the
active site react with the substrate to form products. The products separate
from the enzyme surface, and the enzyme is able to repeat the sequence.
Nonsubstrate molecules that are too bulky or too small alter the shape of the
enzyme so that a misalignment of catalytic groups occurs; such molecules are
not able to react even if they are attracted to the active site.
The induced-fit theory explains a number of anomalous
properties of enzymes. An example is “noncompetitive inhibition,” in which
a compound inhibits the
reaction of an enzyme but does not prevent the binding of the substrate. In
this case, the inhibitor compound attracts the binding group so that the
catalytic group is too far away from the substrate to react. The site at which
the inhibitor binds to the enzyme is not the active site and is called an
allosteric site. The inhibitor changes the shape of the active site to prevent
catalysis without preventing binding of the substrate.
An inhibitor also can distort the active site by affecting
the essential binding group; as a result, the enzyme can no longer attract the
substrate. A so-called activator molecule affects the active site so that a
nonsubstrate molecule is properly aligned and hence can react with the enzyme.
Such activators can affect both binding and catalytic groups at the active
site.
Enzyme flexibility is extremely important because it
provides a mechanism for regulating enzymatic activity. The orientation at the
active site can be disrupted by the binding of an inhibitor at a site other
than the active site. Moreover, the enzyme can be activated by molecules
that induce a
proper alignment of the active site for a substrate that alone cannot induce
this alignment.
As mentioned above, the sites that bind inhibitors and
activators are called allosteric sites to distinguish them from active sites.
Allosteric sites are in fact regulatory sites able to activate or inhibit enzymatic
activity by influencing the shape of the enzyme. When the activator or
inhibitor dissociates from the enzyme, it returns to its normal shape. Thus,
the flexibility of the protein structure allows the operation of a simple,
reversible control
system similar to a thermostat.
Types of allosteric control
Allosteric control can operate in many ways; two examples
serve to illustrate some general effects. A pathway consisting of ten enzymes
is involved in the synthesis of the amino acid histidine. When a cell contains
enough histidine, synthesis stops—an appropriate economy move by the cell.
Synthesis is stopped by the inhibition of the first enzyme in the pathway by
the product, histidine. The inhibition of an enzyme by a product is
called feedback
inhibition; i.e., a product many steps removed from an initial enzyme
blocks its action. Feedback inhibition occurs in many pathways in all living
things.
Allosteric control can also be achieved by activators.
The hormone adrenaline (epinephrine) acts in
this way. When energy is needed, adrenaline is released and activates, by
allosteric activation, the enzyme adenyl cyclase. This enzyme catalyzes a
reaction in which the compound cyclic adenosine monophosphate (cyclic AMP) is
formed from ATP. Cyclic AMP in turn acts as an allosteric activator of enzymes
that speed the metabolism of carbohydrate to
produce energy. This type of allosteric regulation also is widespread in
biological systems. Thus, a combination of allosteric activation and inhibition
allows the production of energy or materials when they are needed and shuts off
production when the supply is adequate.
Allosteric control is a rapid method of regulating products
continuously needed by living things. Yet some cells have no
need for certain enzymes, and it would be wasteful for the cell to synthesize
them. In this case, certain molecules, called repressors,
prevent the synthesis of unneeded enzymes. The repressors are proteins that
bind to DNA and prevent the first step in the process resulting in protein
synthesis. If certain metabolites are added to cells that need an enzyme,
enzyme synthesis occurs—i.e., it is induced. Addition of galactose to
a growth medium containing Escherichia coli bacteria,
for example, induces the
synthesis of the enzyme beta-galactosidase. The bacteria thus can synthesize
this galactose-metabolizing enzyme when it is needed and prevent its synthesis
when it is not. The way in which the synthesis of enzymes is induced or
repressed in mammalian systems is less understood but is believed to be
similar.
Different types of cells in complex organisms have different
enzymes, even though they have the same DNA content. The enzymes
actually synthesized are the ones needed in a specific cell and vary not only
for different types of cells—e.g., nerve, muscle, eye, and skin cells—but also
for different species.
In an enzyme consisting of several subunits, or
chains, alteration in
the shape of one chain as a result of the influence either of a substrate
molecule or of allosteric inhibitors or activators may change the shape of a
neighboring chain. As a result, the binding of a second molecule of substrate
occurs in a different way from the binding of the first, and the third is
different from the second. This phenomenon, called cooperativity, is
characteristic of allosteric enzymes. Cooperativity is reflected by a sigmoid
curve, as compared to the hyperbolic curve of Michaelis–Menten. An enzyme of
several subunits that exhibits cooperativity is far more sensitive to control
mechanisms than is an enzyme of one subunit and hence one active site.
The first example of cooperativity was observed in hemoglobin, which is
not an enzyme but behaves like one in many ways. The absorption of oxygen in the lungs
and its deposition in
the tissues is far more efficient because the subunits of hemoglobin show
positive cooperativity, so called because the first molecule of substrate makes
it easier for the next to bind.
Negative cooperativity, in which the binding of one molecule
makes it less easy for the next to bind, also occurs in living things. Negative
cooperativity makes an enzyme less sensitive to fluctuations in concentrations
of metabolites and may be important for enzymes that must be present in the
cell at relatively constant levels of activity.
Some enzymes are closely associated aggregates of
several enzyme units; the pyruvate dehydrogenase system, for example, contains
five different enzymes, has a total molecular weight of
4,000,000, and consists of four different types of chains. Apparently, the
enzymes in cells may be organized by forming complex units, by being absorbed
on a cell
wall, or by being isolated by membranes in special compartments. Since a
pathway involves the stepwise modification of chemical compounds, aggregations
of the enzymes in a given pathway facilitate their
function in a manner similar to an industrial assembly line.
Discussion
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