The Structure and Function of Enzymes
Chemical reactions in biological systems rarely occur in the absence of a catalyst. These catalysts are specific proteins called enzymes. The striking characteristics of all enzymes are their catalytic power and specificity. Furthermore, the activity of many enzymes is regulated. In addition, some enzymes are intimately involved in the transformation of different forms of energy. Let us examine these highly distinctive and biologically crucial properties of enzymes.
Enzymes Have Enormous Catalytic Power
Enzymes accelerate reactions by factors of at least a million. Indeed, most reactions in biological systems do not occur at perceptible rates in the absence of enzymes. Even a reaction as
simple as the hydration of carbon dioxide is catalyzed by an enzyme.
H 2CO 3CO 2+H 2O
Otherwise, the transfer of CO 2 from the tissues into the blood and then to the alveolar air would be inco
mplete. Carbonic anhydrase, the enzyme that catalyzes this reaction, is one of the fastest known. Each enzyme molecule can hydrate 105 molecules of CO 2 in one second. This catalyzed reaction is 107 times faster than the uncatalyzed reaction.
Enzymes are Highly Specific
Enzymes are highly specific both in the reaction catalyzed and in their choice of reactants, which are called substrates. An enzyme usually catalyzes a single chemical reaction or a set of closely related reactions. The degree of specificity for substrate is usually high and sometimes virtually absolute.
Let us consider proteolytic enzymes as an example. The reaction catalyzed by these enzymes is the hydrolysis of a peptide bond. N H C H
R 1C O N H C H R 2C
O +H 2O Peptide
N H C H
R 1C O -H 3N C H R 2
C O +O -Carboxyl component Amino component
Most proteolytic enzymes also catalyze a different but related reaction, namely the hydrolysis of an ester bond.
R 1C O O 2+H 2
O R 1C O O -+HO R 2+H +
Ester Acid Alcohol
Proteolytic enzymes vary markedly in their degree of substrate specificity. Subtilisin, which comes from certain bacteria, is quite undiscriminating about the nature of the side chains adjacent to the peptide bond to be cleaved. Trypsin is quite specific in that it splits peptide bonds on the carboxyl side of lysine and argentine residues only. Thrombin, an enzyme participating in blood clotting, is even more specific than trypsin. The side chain on the carboxyl side of the susceptible peptide bond must be arginine, whereas the one on the amino side must be glycine.
Another example of the high degree of specificity of enzymes is provided by DNA polymerase I. This enzyme synthesizes DNA by linking together four kinds of nucleotide building blocks. The sequence of nucleotides in the DNA strand that is being synthesized is determined by the sequence of nucleotides in another DNA strand that serves as a template. DNA polymerase I is remarkably precise in carrying out the instructions given by the template. The wrong nucleotide is inserted into a new DAN strand less than once in a million times.
The Activities of Some Enzymes Are Regulated
Some enzymes are synthesized in an inactive precursor form and are activated at a physiologically appropriate time and place. The digestive enzymes exemplify this kind of control. For example, trypsinogen is synthesized in the pancreas and is activated by peptide-bond cleavage in the small intestine to form the active enzyme trypsin. This type of control is also repeatedly used in the sequence of enzymatic reactions leading to the clotting of blood. The enzymatically inactive precursors of proteolytic enzymes are called zymogens.
Another mechanism that controls activity is the covalent insertion of a small group on an enzyme. This control mechanism is called covalent modification. For example, the activities of the enzymes that synt
hesize and degrade glycogen are regulated by the attachment of a phosphoryl group to a specific serine residue on these enzymes. This modification can be reversed by hydrolysis. Specific enzymes catalyze the insertion and removal of phosphoryl and other modifying groups.
A different kind of regulatory mechanism affects many reaction sequences resulting in the synthesis of small molecules such as amino acids. The enzyme that catalyzes the first step in such a biosynthetic pathway is inhibited by the ultimate product. The biosynthesis of isoleucine in bacteria illustrates this type of control, which is called feedback inhibition. Threonine is converted into isoleucine in five steps, the first of which is catalyzed by threonine deaminase. This enzyme is inhibited when the concentration of isoleucine reaches a sufficiently high level. Isoleucine binds to a regulatory site on the enzyme, which is distinct from its catalytic site. The inhibition of threonine deasminase is mediated by an allosteric interaction, which is reversible. When the level of isoleucine drops sufficiently, threonine deaminase becomes active again, and consequently isoleucine is again synthesized.
reaction in the shaftThe specificity of some enzymes is under physiological control. The synthesis of lactose by the mammary gland is a particularly striking example. Lactose synthetase, the enzyme that catalyzes the synthesis of lactose, consists of a catalytic subunit and a modifier subunit. The catalytic subunit by itself cannot synthesize lactose. It has a different role, which is to catalyze the attachment of galactose
to a protein that contains a covalently linked carbohydrate chain. The modifier subunit alters the specificity of the catalytic subunit so that it links galactose to glucose to form lactose. The level of the modifier subunit is under hormonal control. During pregnancy, the catalytic subunit is formed in the mammary gland, but little modifier subunit is formed. At the time of birth, hormonal levels change drastically, and the modifier subunit is synthesized in large amounts. The modifier subunit then binds to the catalytic subunit to form an active lactose synthetase complex that produces large amounts of lactose. This system clearly shows that hormones can exert their physiological effects by altering the specificity of enzymes.
Enzymes Transform Different Kinds of Energy
In many biochemical reactions, the energy of the reactants is converted into a different form with high efficiency. For example, in photosynthesis, light energy is converted into chemical-bond energy. In mitochondria, the free energy contained in small molecules derived from foods is converted into a different currency, that of adenosine triphosphate (A TP). The chemical-bond energy of ATP is them utilized in many different ways. In muscular contraction, the energy of A TP is converted into mechanical energy. Cells and organelles have pumps that utilize ATP to transport molecules and ions against chemical and electrical gradients. These transformations of energy are
carried out by enzyme molecules that are integral parts of highly organized assembilies.
Enzymes Do Not Alter Reaction Equilibria
An enzyme is a catalyst and consequently it cannot alter the equilibrium of a chemical reaction. This means that an enzyme accelerates the forward and reverse reaction by precisely the same factor. Consider the interconversion of A and B. Suppose that in the absence of enzyme the forward rate (K F) is 10-4sec-1and the reverse rate (K R) is 10-6sec-1. The equilibrium constant K is given by the ratio of these rates:
A
-4-1
10-6sec-1
B
K=[B]
[A]
=
K F
K R
=
10-4
10-6
=100
The equilibrium concentration of B is 100 times that of A, whether or not enzyme is present. However, it would take several hours to approach this equilibrium without enzyme, whereas equilibrium would be attained within a second when enzyme is present. Thus, enzymes accelerate the attainment of equilibria but do not shift their positions.
Enzymes Decrease the Activation Energies of Reactions Catalyzed by Them
A chemical reaction, A→B, goes through a transition state that has a higher energy than either A or B. The rate of the forward reaction depends on the temperature and on the difference in free energy between that of A and the transition state, which is called the Gibbs free energy of a activation and symbolized by △G.
∆G≠=G transition state-G substrate
The reaction rate is proportional to the fraction of molecules that have a free energy equal to or greater than △G≠. The proportion of molecules that have an energy equal to or greater than △G≠increases with temperature.
Enzymes accelerate reactions by decreasing △G≠, the activation barrier. The combination of substrate and enzyme creates a new reaction pathway whose transition-state energy is lower than it would be if the reaction were taking place in the absence of enzyme.
Formation of an Enzyme-Substrate Complex Is the First Step in Enzymatic Catalysis The making and breaking of chemical bonds by an enzyme are preceded by the formation of
an enzyme-substrate (ES) complex. The substrate is bound to a specific region of the enzyme called the active site. Most enzymes are highly selective in their binding of substrates. Indeed, the catalytic specificity of enzymes depends in large part on the specificity of the binding process. Furthermore, the control of enzymatic activity may also take place at this stage.
The existence of ES complexes has been shown in a variety of ways:
ES complexes have been directly visualized by electron microscopy and X-ray crystallography. Complexes of nucleic acids and their polymerase enzymes are evident in electron micrographs. Detailed information concerning the location and interactions of glycyl-L-tyrosine, a substrate of carboxypeptidase A, has been obtained from X-ray studies of that ES complex.
The physical properties of an enzyme, such as its solubility or heat stability, frequently change upon for
mation of an ES complex.
The Spectroscopic characteristics of many enzymes and substrates change upon formation of an ES complex just as the absorption spectrum of deoxyhemoglobin changes markedly when it binds oxygen or when it is oxidized to the ferric state, as described previously. These changes are particularly striking if the enzyme contains a colored prosthetic group. Tryptophan synthetase, a bacterial enzyme that contains a pyridoxal phosphate prosthetic group, affords a nice illustration. This enzyme catalyzes the synthesis of L-tryptophan from L-serine and indole. The addition of L-serine to the enzyme produces a marked increase in the fluorescence of the pyridoxal phosphate group. The subsequent addition of indole, the second substrate, quenches this fluorescence to a level lower than that of the enzyme alone. Thus, fluorescence spectroscopy reveals the existence of an enzyme-serine complex and of an enzyme-serine-indole complex. Other spectroscopic techniques, such as nuclear and electron magnetic resonance, also are highly informative about ES interactions.
A high degree of stereospecificity is displayed in the formation of ES complexes. For

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