Biological Chemistry - Berezov, T. T., & Korovkin, B. F. 1998

Enzymes
A Brief History of the Development of Enzymology
Active Site of Enzymes

When studying The Mechanism of an enzyme-catalyzed chemical reaction, researchers are typically concerned not only with identifying intermediate and final products and elucidating individual reaction steps, but also with The Nature of those functional groups within the enzyme molecule that ensure both substrate Specificity and high catalytic activity. In other words, this requires precise knowledge of the geometry and Tertiary Structure of the enzyme, as well as the Chemical Nature of the region(s) within the enzyme molecule responsible for the high velocity of the catalytic reaction. Since substrate Molecules Participating in enzymatic reactions are often quite small compared to enzyme molecules, it was hypothesized that upon The formation of enzyme-substrate complexes, only a restricted portion of The amino acid residues in the peptide chain comes into direct contact with the substrate. This led to METABOLISM/2.html">THE CONCEPT OF the Active Site of an enzyme. The active site refers to a unique combination of amino acid residues in the enzyme molecule that ensures its direct binding to the substrate molecule and direct Participation in the catalytic act (Fig. 4.2). It has been established that in complex Enzymes, prosthetic groups are also part of the active site.

The active site is conventionally divided into the so-called catalytic site, which directly engages in chemical interaction with the substrate, and the binding site, or contact ("anchoring") area, which provides specific affinity for the substrate and the Formation of the enzyme-substrate complex. In turn, the substrate molecule also contains functionally distinct regions: for instance, the substrates of esterases or proteinases feature a single specific bond (or group of atoms) attacked by the enzyme, alongside one or more regions selectively bound by the enzyme.

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Fig. 4.2. The active site of an enzyme (schematic diagram, adapted from Mahler and Cordes).

Dark bands represent Regions of the enzyme polypeptide chain; R denotes amino acid residues and their sequence numbers (starting from the N-terminus).

Experimental evidence has been obtained confirming the presence of two Histidine residues and one Serine residue in the active site of Chymotrypsin, as schematically represented in the three-dimensional structural model of the enzyme precursor (Fig. 4.3). Elucidating the chemical nature and probable Topography of the active site groups is a problem of paramount importance. It boils down to determining the identity of the Amino Acids, their sequence, and their spatial arrangement within the active site. To identify these essential amino acid residues, researchers utilize specific Enzyme Inhibitors (often substrate-like substances or coenzyme analogs), along with Methods of "mild" (limited) Hydrolysis combined with chemical modification, which includes selective oxidation, binding, substitution of amino acid residues, and others.

Fig. 4.3. Hypothetical model of the tertiary STRUCTURE OF THE chymotrypsinogen molecule (adapted from Neurath).

Serine and histidine residues are highlighted in color; the arrow indicates the Cleavage site of the N-terminal region of the polypeptide chain.

Using inhibitory analysis methods, attempts have been made to establish patterns in the Composition and Structure of active sites across various groups of enzymes. Notably, The Use of diisopropylfluorophosphate (DFP), a compound belonging to the class of nerve agents, leads to the complete inactivation of the active site of cholinesterase—the enzyme that catalyzes the hydrolysis of acetylcholine into Choline and acetic acid. It was found that this inhibitor bears a close structural resemblance to acetylcholine and, like the latter, interacts with the OH group of the serine residue in the active site. By inducing phosphorylation of the serine residue in the active sites of several Other Enzymes, DFP likewise inhibits their activity:

It has been demonstrated that DFP selectively phosphorylates only a single functionally active serine residue in each enzyme sensitive to it. Building upon this mechanism of DFP action, attempts have been made to determine the Nature of the amino acids surrounding the "catalytic" serine residue in A number of enzymes (Table 4.2).

As seen from Table 4.2, enzymes with similar modes of action, despite differing in specificity, can exhibit nearly identical Amino acid sequences in the regions flanking the serine residue that bears the functionally active hydroxyl group. Furthermore, the essential role of the serine OH group in catalysis has been proven by chemically blocking or removing it, resulting in the complete loss of enzymatic activity in esterases.

Table 4.2. Amino acid sequences surrounding the serine residue in various esterases and proteinases (adapted from Mahler and Cordes)

It is hypothesized that the formation of an enzyme's active site begins during the early Stages of Protein-enzyme synthesis (see Chapter 14) on the ribosome, when the linear one-dimensional structure of the peptide chain folds into a three-dimensional conformation with a strictly defined architecture. The resulting protein acquires an entirely new type of information, namely functional (specifically, catalytic) information. Any factors leading to Denaturation—i.e., the disruption of tertiary structure—result in the distortion or destruction of the active site structure and, consequently, the loss of the enzyme's catalytic properties. If, under favorable external conditions, the native three-dimensional structure of the enzyme protein can be restored (renatured), its catalytic activity is likewise recovered. This was first demonstrated using pancreatic Ribonuclease as an example (see Fig. 1.13).

In addition to the active site, an enzyme molecule may also feature an allosteric site (or sites) (from the Greek allos — other, different, and steros — spatial, structural). This is a region on the enzyme molecule that binds specific, usually low-molecular-weight substances (effectors or modifiers) whose structures differ from those of the substrates. The binding of an effector to the allosteric site alters the tertiary and often The quaternary structure of the enzyme molecule, thereby modifying the conformation of the active site and leading to either an increase or a decrease in enzymatic activity. Enzymes whose catalytic activity is modulated by allosteric effectors binding to the allosteric site are termed allosteric enzymes*.

Fig. 4.4. Schematic representation of an allosteric enzyme consisting of two protomers joined by heterologous ("HEAD-to-tail") association (adapted from Koshland).

S — substrate; M₁ — modifier binding at the active site; M₂ — modifier binding at the allosteric site (effector).

A distinctive feature of many allosteric enzymes is the presence of multiple active sites and multiple allosteric regulatory sites within an oligomeric enzyme molecule, spatially separated from one another. In an allosteric enzyme, each of the two symmetrically arranged protomers contains one active site that binds substrate S and one allosteric site that binds effector M₂, totaling 2 sites per enzyme molecule (Fig. 4.4). Evidence indicates that toward the substrate, allosteric enzymes possess not only active sites but also so-called effector sites; upon binding to an effector site, the substrate does not undergo catalytic conversion, yet it influences the catalytic efficiency of the active site. Such interactions between sites binding ligands of the same type are referred to as homotropic interactions, whereas interactions between sites binding Different types of ligands are termed heterotropic interactions.

Thus, the gathered data concerning the chemical nature of the active site and allosteric regions indicate that Enzymatic Catalysis, much like the substrate-binding reaction, involves not a restricted and minor portion of the enzyme—as was previously assumed—but a significantly larger part of the enzyme protein molecule. These circumstances most likely explain the large size and bulkiness of the enzyme's three-dimensional molecular structure; the same factors must be taken into account when designing artificial low-molecular-weight enzyme analogs (synzymes) that possess The properties of native enzymes (see above).

* Some authors recommend using the term "regulatory center" (regulatory enzyme) for enzymes endowed with regulatory Functions, since this supposedly eliminates the need to specify the presence of a distinct effector-binding site on the enzyme's surface.