Fundamentals of Biochemistry - A. A. Anisimov 1986

Enzymes
Active site

During enzymatic reactions, contact is established between the enzyme and the substrate, resulting in The formation of intermediate enzyme-substrate complexes (ES). The region of the enzyme molecule where substrate binding and transformation take place is called the active center (usually accounting for only a small fraction of the entire molecule). The active center is formed by specific side chains of amino acid residues in the polypeptide chain, while in two-component Enzymes, it also includes certain non-protein moieties. In enzymes with a quaternary Structure, the number of active centers generally corresponds to the number of subunits.

The active center is functionally heterogeneous and can be conventionally divided into several zones. The groups of the active center that come into contact with the reactive fragments of the substrate molecules—meaning they take direct part in the synthesis or Cleavage of a substrate bond—comprise the catalytic zone. The groups that interact with the non-reactive fragments of the substrate and anchor it within the active center belong to the binding zone (Fig. 3.1).

Substrate binding is typically multipoint, occurring through the participation of several groups from both the enzyme molecule and the substrate. The enzyme molecule also contains amino acid residues that do not make direct contact with the substrate, but nevertheless facilitate catalysis by holding the groups of the catalytic zone in the proper active conformation.

Most commonly, active centers are composed of Amino Acids such as Ser, His, Thr, Cys, Glu, Asp, and Arg. The amino acids that form the active center are located far apart along the primary polypeptide chain, but are brought into close proximity when the three-dimensional structure is formed. For example, the active center of Chymotrypsin includes His-57, Asp-102, and Ser-195, out of a total of 246 amino acid residues in the enzyme. Chymotrypsin, like several other predominantly secreted enzymes, is initially synthesized in an inactive form known as a proenzyme (zymogen)—chymotrypsinogen. Chymotrypsinogen lacks a fully formed active center and is incapable of efficiently catalyzing the reactions typical of the active enzyme. Upon the activation of chymotrypsinogen by Trypsin and chymotrypsin, four peptide bonds are hydrolyzed, releasing dipeptides 14–15 and 147–148 (Fig. 3.2), which completes the Formation of the active center and brings amino acids 57, 102, and 195—directly involved in catalysis—into close proximity (see Fig. 3.7).

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Fig. 3.1. The active center of an enzyme:

a — groups of the catalytic zone, b — groups of the binding zone

Fig. 3.2. Activation of chymotrypsinogen (single polypeptide chain).

The resulting δ-chymotrypsin consists of three chains linked by Disulfide Bonds

For many enzymes, the specific amino acids that constitute the active center and their catalytic roles are now well established. Conclusions regarding the presence of particular amino acid residues of the enzyme molecule within the active center can be drawn by studying the Kinetics of Enzymatic reactions and using specific group Reagents. Specific reagents have been identified for amino acids such as Trp (N-Bromosuccinimide), Cys (organic mercury derivatives, e.g., p-chloromercuribenzoate — PCMB, monoiodoacetic acid), Ser (diisopropyl fluorophosphate — DFP), and several others.

Information about the Functional groups of the active center can also be obtained through The Use of Proteolytic Enzymes that selectively cleave peptide bonds in the enzyme molecule. Valuable insights are provided by comparing the three-dimensional structures of the enzyme in the absence and presence of inhibitors, combined with Primary Structure data.

The active centers of enzymes are typically located in clefts or depressions On the surface of the enzyme molecule (Fig. 3.3). For instance, in many dehydrogenases, the active centers reside in a cleft between two domains. The microenvironment of the active center differs from the rest of the enzyme's surroundings by having a lower dielectric constant, approaching that of certain organic Solvents. An environment with a reduced dielectric constant is more favorable than Water for charge-transfer reactions, which are characteristic of the action of numerous enzymes. In addition to this feature, the microenvironment of the active center is characterized by increased microviscosity, which restricts the rotational freedom of the active center groups. If an enzyme is two-component, its active center is completed only after the interaction of the apoenzyme with the non-protein moiety. It has been shown, for example, that Pyruvate—the substrate for the enzyme Lactate dehydrogenase—is not bound by the enzyme until a complex is formed between the apoenzyme and NADH. Consequently, the pyruvate-binding site on the enzyme surface emerges only after NADH binding, driven by a conformational change and possibly a local alteration in the charge of the protein molecule.

Fig. 3.3. Model of an enzyme molecule. A — Tertiary Structure of the molecule; B — molecular silhouette showing the active center (boxed)

The individual Structural Features of the active centers in different enzymes determine their catalytic Specificity. Enzyme Specificity can be absolute or relative. In the case of absolute specificity, enzymes catalyze the transformation of only a single substance, whereas in relative specificity, they act on a narrow group of closely related compounds. Examples of enzymes with absolute specificity include urease and succinate dehydrogenase (SDH):

Enzymes with relative specificity include esterases, which cleave a wide range of carboxylic acid esters; certain Phosphatases acting on phosphoric acid esters; and peptidases and proteinases, which hydrolyze Peptides and Proteins.

The relative specificity of proteinases is also manifested in their ability to cleave A large number of different proteins. In some enzymes, this is combined with The ability to hydrolyze only specific peptide bonds within the substrate, which is governed by the structural features of their active centers. For example, differences in the catalytic selectivity of trypsin, chymotrypsin, and Elastase stem from minor structural variations in the region of the active center that accommodates the side chain of the cleaved substrate's amino acid.

In chymotrypsin, this region (the "pocket") is adapted to bind large hydrophobic radicals (Phe, Trp, Tyr), whereas in trypsin, it binds positively charged groups because a negatively charged COO^- group of Asp-189 is located at the bottom of the active center pocket. In elastase (which hydrolyzes proteins at Ala residues), the entrance to the pocket features Val and Thr instead of the two Gly residues found in chymotrypsin, thereby preventing large substrate side chains from entering.

Similar principles explain the differences in specificity between Carboxypeptidases A and B. Both enzymes hydrolyze proteins from the C-terminus: the former most rapidly cleaves C-terminal aromatic amino acids (Tyr, Trp, Phe), while the latter preferentially cleaves Lys and Arg. The primary difference between these enzymes lies in the binding residue within the pocket—Ile-255 in Carboxypeptidase A versus Asp-255 in carboxypeptidase B—which dictates their affinity for different C-terminal amino acid residues.

In addition to chemical specificity, some enzymes exhibit steric specificity. Such enzymes can distinguish between stereoisomers and catalyze the transformation of only one of them: D- or L-, α- or β-, cis- or trans-. For instance, there are α- and β-glycosidases that hydrolyze only one specific type of glycoside. Fumarate hydratase acts exclusively on fumarate (the trans-isomer) and does not convert maleate (the cis-isomer). Conversely, during the dehydration of malate (a reaction occurring in The Tricarboxylic Acid Cycle), only Fumarate is formed: