The Mechanism of the α‐Chymotrypsin and Trypsin‐Catalyzed Hydrolysis of Amides

Abstract

The applicability of the acylenzyme mechanism to the tryptic hydrolysis of the amide bond has been studied. It was found that trypsin acetylated a t the hydroxyl group of the "active" serine or acetyl (Ser-183)trypsin displays no activity with respect to both esters (N-benzoyl-L-arginine ethyl ester and p-nitrophenyl-p'-guanidinobenzoate) and anilides (N-carbobenzoxy-L-arginine m-nitroanilide and N-benzoyl-DL-arginine p-nitroanilide). It was also found, that deacetylation of acetyl(Ser-183)trypsin is effectively promoted by N-carbobenzoxy-L-arginine m-nitroanilide (Z-Arg-Nan) taken a t a high concentration. As was found by means of a detailed kinetic analysis of experimental results, the binding of this anilide with acetyl(Ser-183)trypsin increases the rate constant of deacylation of the latter by as many as 23 times. This effect which results in a rapid increase in the concentration of native trypsin in the reaction system, causes a marked increase in both kcat and gm(agg) of tryptic hydrolysis of Z-Arg-Nan). Apparently, the phenomena is the main reason for the erroneous conclusions of Bresler et al. (1969, 1971), who stated : trypsin with an acylated active serine remains a highly efficient catalyst of amide hydrolysis. Finally, the data of Bresler et al. on tryptic hydrolysis and hydroxylaminolysis which contradict the acylenzyme mechanism of hydrolysis of the amide bond can be readily accounted for by chemistry of hydroxylamine, as was done by Bender et al. Epand andWilson (1963).Thus, the data obtained so far strongly support the acyl-enzyme mechanism of the hydrolysis of the amide bond catalyzed by trypsin and a-chymotrypsin.I n the late forties, a-chymotrypsin and trypsin were established to have, in addition to the amidase and peptidase activities, an esterase activity. Since then the question has arisen whether the enzymic hydrolysis of amide and esteric substrates proceeds via a similar mechanism. The problem was originally posed in a more general way: does the hydrolysis of esters and amides take place on one active site of the enzyme or are the esterase and amidase activities controlled each by a centre of its own. The first papers dealing with this problem appeared as early as 1949 [1,2]. I n these and subsequent papers Jansen and Balls with co-workers, as well as other authors (see [3]), showed that the phosphorylation of a-chymotrypsin or trypsin is accompanied by a simultaneous decrease in the esterase, amidase and peptidase activities. The fact of the existence of the common active site in a-chymotrypsin was also supported by the data on photooxidation of the enzyme [4] Abbreviations. Z-Arg-Nan, N-carboxbenzoxy-L-arginine m-nitroanilide ; Bz-Arg-Nan, N-benzoyl-DL-arginine p-nitroanilide; Bz-Arg-OEt, N-benzoyl-L-arginine ethyl ester.Enzyme. Trypsin (EC 3.4.4.4).35 Eur. J. Biochem., Vo1.38 in which a-chymotrypsin inactivates with respect to both esteric and amide substrates. However, these data only suggest that a-chymotryptic and tryptic hydrolyses of amide and esteric substrates occur on the ...

Synthesis of Cytarabine Lipid Drug Conjugate for Treatment of Meningeal Leukemia: Development, Characterization and <I>In</I> <I>Vitro</I> Cell Line Studies

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Mutation of Residue βF71 of Escherichia coli Penicillin Acylase Results in Enhanced Enantioselectivity and Improved Catalytic Properties

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