Enzymatic Catalysis

the acceleration of chemical reactions through the action of enzymes. Chemical reactions lie at the base of an organism’s vital activities, breaking down food substances, producing necessary organic compounds by synthesis, and converting the energy of these compounds into energy for physiological processes (operation of muscles, kidneys, nervous system). None of these reactions could proceed with the speed required by the organism if the mechanisms for acceleration through enzymatic catalysis had not appeared during evolution.

It was once thought that enzymatic catalysis was qualitatively different from the nonbiological catalysis widely used in industry. This notion was based on three distinguishing features of enzymatic catalysis. The first is the exceptionally high efficiency, with reaction rates being increased by a factor between 10¹⁰ and 10¹³; the second is the specificity, that is, the ability of each enzyme to select a narrow group of substrates and sometimes to catalyze the conversion of only a single substance in a single direction. The third feature is the control of enzyme activity, that is, the ability of the biocatalyst, or enzyme, to increase or decrease its own activity depending on the organism’s requirements. However, investigation of the mechanism of enzymatic catalysis shows that the laws and principles governing conventional chemical reactions are applicable. The reactions in enzymatic catalysis, however, are distinguished by the complexity of the chemical conversions and enzyme structures.

The efficiency of enzymatic catalysis is attained through the breaking down of the chemical reaction into a series of energetically simpler intermediate reactions in which enzymes participate. The most important reaction in enzymatic catalysis is the formation of a primary enzyme-substrate complex that provides a gain in energy sufficient to accelerate the whole process. The necessity of such a complex was suggested by studies showing the velocity (V) of the enzymatic reaction to be dependent on the concentrations of the enzyme (E) and substrate (S); the velocity is expressed by the Michaelis-Menten equation:

Here, k₃ and K_m are constants characteristic of each reaction.

This relationship, which has been experimentally confirmed for many enzymatic reactions, can be theoretically deduced if the conversion of the substrate into the reaction product (P) occurs through a mechanism for the formation and breakdown of a complex comprising the enzyme and substrate, that is, an ES complex:

Here, k₁, k_–1, and k₊₂ are constants characteristic of the speed of the stage of the process indicated by arrows, the relationship (k_–1 + k₊₂)/k₊₁ being equal to K_m. If not one but several substrates participate in the reaction (two substrates figuring in the majority of cases) and the ES complex forms reaction products in not one but several stages, the relationship is expressed by more complex equations; however, these equations too can be derived only by presupposing the prior formation of ES complexes. For many enzymes, direct proof of the formation of ES complexes has been found. Thus, the formation of complexes incorporating dehydrogenases and peroxidases has been proved by spectral methods, and complexes of the oxidase of D-amino acids with D-alanine and of carboxypeptidase A with glycyl-L-tyrosine have been isolated in the crystalline state. In a number of cases, the three-dimensional structure of ES complexes has been established through X-ray diffraction analysis.

The high specificity of enzymatic catalysis is explained by the strict geometric and electronic correspondence between the structure of the substrate and that of the enzyme’s active site, where the sorbed substrate undergoes further chemical changes. It has been postulated that the correspondence (complementarity) between the geometric and electronic structure of the active site and that of the part of the molecule of the substrate(s) reacting with the site is achieved as the substrate approaches the enzyme surface (the induced-fit theory of D. E. Koshland, USA). The enzyme’s active site, which is an assembly of chemically active groups (functional groups of amino acids), is formed from amino acid residues often at some distance from one another in the polypeptide chain but brought into proximity as a result of the three-dimensional structure of the protein. Substances of low molecular weight (metal ions, organic cofactors) often figure in the makeup of the active sites. In the α-chymotrypsin molecule, which catalyzes the hydrolysis of proteins and polypeptides and which has a chain of 246 amino acid residues, the active site is formed of residues of serine (the chain’s 195th residue), histidine (57th), isoleucine (16th) and aspartic acid (102nd and 194th). The active center of ribonuclease, which catalyzes the breakdown of ribonucleic acid, comprises 124 amino acids. It is made up of residues of lysine (seventh and 41st), arginine (39th), and histidine (12th and 119th). The active sites of many enzymes function together with substances of low molecular weight—the cofactors of enzymatic catalysis. Vitamin derivatives and coenzymes are among the cofactors, as are the ions of certain metals (Na, K, Ca, Mg, Zn, Fe, Cu, Co, Mo).

Although a general theory of enzymatic catalysis does not exist, research on the mechanism of enzyme activity permits a qualitative and sometimes a quantitative explanation of the high activity of the catalysis. The principal causes of this activity are (1) the proximity of the reactants at the time of sorption onto the active site, a factor equivalent to raising the reactants’ concentrations; (2) the particular orientation of the substrate sorbed onto the active site, which favors interaction with the site’s catalytic segment; (3) the formation of chemical bonds between the substrate and the catalytic segment of the active site, a process directing the reaction along the pathway that is energetically easiest; (4) the execution of the basic chemical conversions intramolecularly, that is, within the enzyme-substrate complex; and (5) the exceptional flexibility of the enzyme molecule, allowing the active site to assume at each stage of the conversion of the enzyme-substrate complex the structure that helps the stage to proceed at the maximum speed. Each stage creates the optimal conditions for the subsequent stage. A rough evaluation of the overall effect of the factors in enzymatic catalysis listed above makes it possible to predict theoretically that the rate of a reaction will be accelerated by a factor of 10¹⁰ to 10^–3, a figure that in many cases coincides with values obtained experimentally.

The mechanisms for controlling the activity of enzymes derive from features of the enzymes’ protein structure. The three-dimensional structure of enzymes, which is maintained by relatively weak chemical bonds between different segments of the polypeptide chain, is easily disrupted by changes in a cell’s temperature, acidity, and salt concentration. Since catalysis requires that the enzyme have a specific structure, all these factors have an effect on activity. Each enzyme is activated to its maximum extent at a certain temperature and pH. Departure from the optimum conditions in either direction reduces enzyme activity; often, the activity is regulated by the product of the reaction. For reversible processes in which an enzyme catalyzes the forward and reverse reactions, the rate of the forward reaction (the activity of enzymatic catalysis) is reduced when an excess of the reaction product is formed.

Allosteric control plays an important role in enzymatic catalysis. A great number of sequential chemical reactions, each catalyzed by a specific enzyme (E₁, E₂,..., E_n), occur in the living cell:

Numerous reactions have been observed in which the product P, when formed in excess of physiologically necessary quantities, displays the ability to reduce the activity of the first enzyme E₁ and thereby to reduce the rate of the whole chain of reactions. Such a mechanism is called feedback inhibition. Here, the inhibitor P (referred to in general as an effector) acts on the enzyme E₁, at its own special site of attachment, which is at some distance from the active site. However, owing to the mobility of the enzyme’s protein structure, the attachment of the inhibitor leads to a change in the structure and properties of the active site. F. Jacob and J. Monod have proposed that this site be called the allosteric site and that enzymes of the E₁ type be called allosteric enzymes. Nucleotides, for example, adenylic acid and adenosine triphosphate, and amino acids figuring in the reactions for the biosynthesis of other amino acids function as allosteric effectors.

Also considered allosteric are mechanisms controlling the activity of enzymes with several active sites. Here, the attachment of the substrate to the active site causes either a decrease or an increase in the activity of the enzyme. Enzymes constructed of several (an even number) molecules, each of which has an active site and an allosteric site, possess allosteric properties. The action of the effector at the allosteric site of one of the molecules is such as to change (cooperative interaction) the structure of the other molecules and the activity of the enzyme as a whole. Regulatory mechanisms are also possible in which the influence of the effector on an allosteric enzyme leads to a change in the level of association of the enzyme’s subunits, a process accompanied by a change in the overall activity of the enzyme. Mechanisms of this type play an important role in regulating complex systems of chemical reactions (metabolism) in organisms.