Principles of Protein Structure - G. Schultz 1982

Structural Basis of Protein Mechanism, Action, and Function
Enzymatic Catalysis
Catalytic Mechanism of Chymotrypsin

The catalytic Mechanism of Chymotrypsin, a peptide bond-cleaving enzyme, has been studied in greater detail than that of any other enzyme [537]. Such investigations are facilitated by several Characteristic Features of chymotrypsin. It is a monomeric enzyme that displays no allosteric effects; the structural changes accompanying peptide bond Cleavage are very small; and finally, chymotrypsin has The ability to transfer acyl groups from A wide variety of Donors (such as Peptides and esters) to an equally diverse range of acceptors, such as Water, alcohols, or amines. The ability to compare processes involving different donors and acceptors greatly simplifies the analysis of individual catalytic steps [733, 734].

Based on X-Ray Diffraction, spectroscopic, and chemical data (reviewed in [537]), a plausible mechanism of chymotrypsin action has been proposed (Fig. 11.1). After the enzyme and substrate form a Michaelis complex (Section 10.2), the oxygen atom of the hydroxyl group of the Ser-195 residue attacks the carbonyl carbon atom of the labile substrate bond, yielding an unstable tetrahedral intermediate [735]. This reaction is facilitated by a charge-Relay system [628, 736], which transfers a proton from the hydroxyl group of Ser-195, thereby converting it into a potent nucleophile. Next, His-57 donates a proton to the nitrogen atom of the cleavable peptide bond, resulting in its cleavage. At this stage, the amine moiety forms a Hydrogen bond with the His-57 residue, while the acyl group becomes attached to Ser-195 via an ester linkage. This completes the acylation phase of the hydrolytic reaction.

The second half of the process is deacylation. The amine portion of the substrate departs, and a water molecule takes its place in the Active Site. In principle, deacylation is the reverse of acylation, with H₂O replacing the amine component [85]. Initially, the charge-relay system abstracts a proton from the water molecule, and the resulting OH^- ion simultaneously attacks the carbonyl carbon of the acyl group attached to Ser-195. This generates an unstable tetrahedral adduct in which His-57 donates a proton to the oxygen atom of the Ser-195 residue, releasing the acidic component of the substrate, which then diffuses away. The enzyme is now ready for the next catalytic cycle.

Chymotrypsin, glyceraldehyde-3-phosphate dehydrogenase, and Papain employ analogous reaction mechanisms. The mechanism described above is characteristic not only of phylogenetic relatives of chymotrypsin, such as Trypsin or Thrombin, but is also utilized by other Protein Families. For instance, a step-by-step analogy has been noted in the Reactions Catalyzed by such diverse Enzymes as chymotrypsin, papain, and glyceraldehyde-3-phosphate dehydrogenase [737] (Fig. 11.1).

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Fig. 11.1. Analogy between the catalytic mechanisms of chymotrypsin, papain, and glyceraldehyde-3-phosphate dehydrogenase [737].

The first Column lists the individual stages common to all three enzymes. The tetrahedral intermediates (tetrahedral adducts) presumably represent transition states. The Asp-His-Ser charge-relay system of chymotrypsin is shown on the left. For the substrate, only the cleavable peptide bond R₁—NH—CO—R₂ is depicted. The peptide chain runs from right to left, as is customary in protease mechanism diagrams. All Stages of the chymotrypsin-catalyzed reaction are discussed in the text; the second tetrahedral intermediate mentioned there is shown in the figure only in its initial phase, where H₂O attacks the acyl intermediate. Papain is a so-called sulfhydryl protease, in which an SH group serves as the nucleophilic counterpart to the OH group of the Ser-195 residue in chymotrypsin. The enzyme shown in the fourth column catalyzes the reaction: glyceraldehyde-3-phosphate + NAD + inorganic phosphate ⇄ 1,3-diphosphoglycerate + NADH (substrates shown are O=CHR, the nicotinamide ring, and HPO^2-₄). In this case, the process leading from the tetrahedral adduct to the acyl-enzyme is a hydride ion elimination reaction, i.e., an oxidation step. The acyl intermediate is attacked not by an H₂O molecule, as in both proteases, but by inorganic phosphate, so that the final reaction product is an acid anhydride.