Biochemical Engineering Fundamentals, Part 1 - Bailey J., Ollis D. 1989

Kinetics of Enzyme-Catalyzed Reactions
Enzyme-Substrate Complexes and the Mechanism of Enzyme Action

Currently, there is no single theory that explains the unusually high Specificity and activity of enzymatic catalysts. At the same time, for a small number of specific Enzymes, a variety of highly plausible hypotheses supported by experimental data have been proposed. Apparently, the phenomena underlying these hypotheses collectively account for the specific METABOLISM/8.html">Properties of Enzymes. In this section, we will briefly review some of these hypotheses; more detailed information can be found in the literature cited at the end of the chapter. Since all the hypotheses considered here only partially explain the Mechanism of enzyme Action, we will not attempt to generalize these data to formulate a unified theory of enzymatic activity.

The existence of enzyme-substrate complexes has been proven using various experimental Methods, including X-ray crystallography, spectroscopic techniques, and electron paramagnetic Resonance. The substrate binds to the enzyme at a specific region of the enzyme molecule called the Active Site, where the enzyme-catalyzed reaction occurs and its products are formed. Weak interactions, discussed in Section 2.4.3, sometimes participate in substrate-enzyme binding and complex formation, and in some cases, covalent bonds are also formed. As shown schematically in Figs. 2.24 and 3.2, the complex is formed when the substrate "key" fits into the enzyme "lock". Fig. 3.2 shows particularly clearly that the enzyme-substrate complex is formed via Hydrogen Bonds between the substrate and groups located in widely different PARTS OF THE enzyme's Amino Acid Sequence.

This example also clearly illustrates THE CONCEPT OF the active site. The protein molecule is folded in such a way that the reactive groups in the side chains of several amino acid residues of the enzyme form a highly specific, spatially organized Structure that precisely matches the configuration of the substrate. The active sites of enzymes include the side chains of Asp, Cys, Glu, His, Lys, Met, Ser, and Thr residues, as well as terminal amino and carboxyl groups. Since on average about 20 such groups are located near the substrate (significantly fewer than the total number of amino acid residues in the enzyme molecule), it is generally accepted that only a small part of the enzyme is directly involved in the functioning of its active site. Large enzymes may have multiple active sites. Most of The amino acid residues not included in the active site determine the folding pattern of the polypeptide chain (Secondary structure) and the spatial arrangement of one part of the chain relative to another (tertiary structure), which ultimately creates the Active Site of the enzyme (Fig. 3.4).

Although some of the more detailed hypotheses presented below are still not without certain contradictions, it should be emphasized that The concepts of the active site and the enzyme-substrate complex are currently generally accepted and form The basis of most theories explaining The Mechanism of enzyme action. In the following sections, we will also use these concepts in the mathematical analysis of enzyme catalysis kinetics.

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FIG. 3.2. Schematic representation of the active site of Lysozyme. The hexasaccharide substrate molecule is highlighted with bold lines, large and small circles denote oxygen and nitrogen atoms, respectively, and dashed lines represent hydrogen bonds. [Reproduced with permission from: Yudkin M., Offord R., A Guidebook to Biochemistry, p. 48, Cambridge University Press, London, 1971.]

Two different approaches to explaining enzymatic activity are shown schematically in Fig. 3.3. An enzyme can bind two substrate molecules in such a way that their reactive groups are positioned close to each other and to the catalytic groups of the enzyme. This naturally facilitates the acceleration of the chemical reaction; this effect is known as the proximity effect. Suppose further that the molecules of the two substrates do not possess spherical Symmetry. In this case, the reaction will occur only if, upon coming together, the molecules are aligned in an orientation that ensures close interaction of the reactive atoms or groups. It is believed that enzymes bind substrate molecules so that the latter occupy a particularly favorable position, thereby creating an orientation effect that accelerates the reaction. This phenomenon, sometimes also referred to as orbital steering, contributes to The process of Enzymatic Catalysis, although the quantitative magnitude of the corresponding effect is generally still difficult to determine.

FIG. 3.3. An enzyme accelerates a reaction by bringing two substrates into close spatial proximity (proximity effect) and aligning them at a favorable angle (orientation effect). (Reproduced with permission of D. E. Koshland.)

Before proceeding to a Brief Overview of the chemistry of enzymatic catalysis, another hypothesis related to enzyme geometry should be mentioned. It is known that substrate binding is accompanied by a slight change in the Spatial Structure of some enzymes. In particular, by studying the three-dimensional STRUCTURE OF THE enzymes lysozyme and Carboxypeptidase A in the form of complexes with and without substrates, it was shown that the Conformations of the enzymes change slightly upon substrate binding. Such induced fit of the enzyme and substrate may contribute to the process of enzymatic catalysis. More sophisticated Variants of the induced-fit model have also been proposed, in which several intermediate enzyme-substrate complexes are formed sequentially during the process. Some elasticity and flexibility of the enzyme molecule can facilitate the precise spatial alignment of its catalytic groups, thereby accelerating The conversion of each intermediate. It is possible that substrate-induced Changes in the conformation of the active site are characteristic of enzymatic catalysis in general, although the experimental detection of this effect is associated with A number of difficulties and has therefore been achieved for only a few enzymes.

Some enzymes carry out reactions well known to the organic chemist. One such reaction is general Acid-Base Catalysis, in which the catalyst accepts or donates a proton at one stage of the process. This type of catalysis, in particular, underlies the MECHANISM OF ACTION of one of the few enzymes for which a sufficiently convincing complete sequence of elementary steps of the catalytic process has been proposed (Fig. 3.4). Chymotrypsin, isolated from the Pancreas, is a proteolytic (i.e., protein-hydrolyzing) enzyme that specifically cleaves peptide bonds formed by the carboxyl groups of Tyrosine, Tryptophan, and phenylalanine residues. In the chymotrypsin-catalyzed reaction, Water is believed to act as the carrier for both proton abstraction and addition. In essence, a number of reactions highly important in Cell chemistry, including addition to carbonyl compounds and ester Hydrolysis, can be reduced to general acid-base catalysis.

Other factors may also play an important role in enzymatic catalysis, such as Covalent Catalysis, bond strain, electrostatic catalysis, multifunctional catalysis, and solvent effects (recall the oil drop structure described in the previous chapter). A detailed description of these effects can be found in the literature cited at the end of the chapter; like the factors discussed in this section, they likely influence some enzyme-catalyzed reactions. Since enzymatic catalysis reactions generally involve multiple effects, it is not surprising that a simple general scheme to evaluate their combined impact and the relative importance of each has not yet been developed. Fortunately, mathematical expressions for the rates of enzyme-catalyzed reactions can be derived based solely on the fundamental Concept of the enzyme-substrate complex as the primary intermediate.

FIG. 3.4. a — Proposed reaction sequence for the Cleavage of a peptide bond by chymotrypsin. (Reproduced from: Lehninger A. L., Biochemistry, 2nd ed., pp. 229—231, Worth Publishers, New York, 1975); b — Structure of chymotrypsin. [Reproduced from: Matthews B. W. et al., Three-Dimensional Structure of Tosyl-a-Chymotrypsin; Nature, 214, 652 (1976)].

A. — The hydroxyl group of Ser195 is hydrogen-bonded to the imidazole nitrogen atom of His 57.

B. A proton migrates very rapidly from Ser195 to the imidazole nitrogen of His57. A transient Hydrogen bond is formed between the imidazole ring and the substrate, orienting the dipeptide in such a way that nucleophilic attack of the Serine oxygen atom on the carbonyl carbon of the substrate becomes possible.

C. The aminoacyl group is cleaved from the dipeptide and transferred to the enzyme; simultaneously, the free C-terminal amino acid of the substrate is formed.

D. Under the action of H₂O, the aminoacyl group is cleaved from Ser195, thereby regenerating the free enzyme.