Mechanism of chymotrypsin. Structure, reactivity, and nonproductive binding relations

Abstract

It is shown that anilide substrates of chymotrypsin may bind in a nonproductive mode, with the anilide ring in the substrate binding pocket. Acetyl-and trimethylammoniumsubstituted TV-acetyl-u-tyrosineanilides bind predominantly productively. Hydrophobic substituents such as -Cl and -NO% groups cause nonproductive binding to be 50-100 times stronger than productive. The tight binding of these hydrophobic substrates is not due to the accumulation of a covalent intermediate. • Similarly, the lowered ¿cat values are not due to electronic effects on chemical reactivity but are a consequence of the nonproductive binding. At pH 7.8 the dissociation constants of V-acetyl-L-tyrosine-4-chloroanilide, V-acetylglycine-4-chloroanilide, and formyl-4-chloroanilide are 0.78, 0.98, and 0.40 mM, respectively. The binding is almost completely associated with that of the 4-chloroanilide portion. The dissociation constants of a series of substituted formanilides follow 7r, the hydrophobicity constant. The binding of substituted A-acetyltyrosineanilides can be separated into the T A he chymotrypsin-catalyzed hydrolysis of specific anilide substrates is of some mechanistic and historical importance.Substituents in the aniline ring cause inductive effects which alter chemical reactivity but, it would be thought, cause only small steric effects. The hydrolysis of a series of substituted anilides of TV-acetyl-L-tyrosine and V-acetyl-L-tryptophan has led to three theories of the mechanism of action of chymotrypsin based on the experimental linear free-energy relationships.It was found that for a limited series of substituted anilides both ¿cat* 1 **and Km are decreased by electron withdrawal and that the apparent pKa for the enzyme-substrate complex is in some cases significantly lowered (Inagami et al., 1969). These authors suggested the possible contribution of an acidic group in the catalysis. Wang and coworkers (Wang and Parker, 1968; Parker and Wang, 1969) have formulated a general theory of enzyme catalysis by "directed proton transfers" based on the variation of ¿cat with aniline basic strength. Caplow (1969) and Lucas and Caplow (1972), on the basis of the variation of ¿cat, Km, and the of ¿cat with substituents, postulated the accumulation of a tetrahedral intermediate in the reaction-the enzyme stabilizing the otherwise unstable intermediate.

Modulation of the general anesthetic sensitivity of a protein: a transition between two forms of firefly luciferase.

“…These values are large even compared to that for the transfer of methylene groups from water to the completely apolar solvent hexadecane (9). Other examples of "enhanced hydrophobicity" have been reported, but for enzymes such as chymotrypsin (10,11) and certain tRNA synthetases (12) Our results also have important implications for the cutoff effect in general anesthesia: as one ascends a homologous series of anesthetic agents, such as the primary alcohols, potencies increase until a point is reached above which higher members of the series are inactive. The cutoff point for primary alcohols is just after C12 for tadpoles; i.e., 1-tridecanol does not cause anesthesia (7).…”

Acylation of α-Chymotrypsin by Oxygen and Sulfur Esters of Specific Substrates: Kinetic Evidence for a Tetrahedral Intermediate

“…Some of the first evidence for such intermediates in nonenzymatic acyl-transfer reactions came from the study of the alkaline hydrolysis of esters labeled specifically in the carbonyl group with 180 (1). The occurrence of a similar intermediate in the a-chymotrypsin-catalyzed hydrolysis of some anilides and hydrazides has been recently proposed from kinetic studies by several research groups (2)(3)(4)(5)(6)(7)(8)(9). An important contribution to this problem on (nonspecific) ester substrates has been made by Frankfater and K6zdy (10), who obtained identical rate constants for the a-chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate and thiolacetate.…”

Targeted rescue of a destabilized mutant of p53 by an in silico screened drug

“…Small induced-fit structural changes bedevil in silico screening methods, and so it is important to have solved the structure of the complex. Even small changes in the structures of substrates and ligands have been known from early studies to cause radical changes in modes of binding (27).…”