Catalytic Activity of Trypsin with an Acylated “Active” Serine

Bresler, Simon E.; Krutyakov, Valerii M.; Vlasov, G. P.

doi:10.1111/j.1432-1033.1971.tb01223.x

Cited by 5 publications

(8 citation statements)

References 20 publications

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“…The reasons for the discrepancy between my results and the conclusion reached by Bresler et al [2] are not clear. The use of the acetyl moiety as a serine blocking agent makes possible some experimental artifacts due to deacylation which could render interpretation of their data difficult.…”

Section: Discussioncontrasting

confidence: 96%

“…There is no suggestion that the carbamylated enzyme may still be able to catalyze hydrolysis of the anilide by some mechanism not involving an acyl-enzyme intermediate. This woull seem to directly contradict the conclusions of Bresler et al [2].…”

Section: Resultsmentioning

confidence: 80%

“…Bresler, Krutyakov and Vlasov [2] studied the relative activities towards ester and anilide substrates of trypsin in which the active site serine was acylated with various small aliphatic acids. Using the acetyl derivative, for instance, they found 6% remained of the activity of unmodified trypsin with N"-carbobenzoxy-L-arginine ethyl ester as the substrate, while with N"-carbobenzoxy-L-arginine-m-nitroanilide as the substrate they found that 37% of the initial activity remained.…”

Section: Discussionmentioning

confidence: 99%

“…It is known that small molecules can accelerate the deacylation of acetylchymotrypsin [9,IO]. Bresler et al have apparently considered this possibility and stated that they carried out control experiments which ruled it out [2]. It is 46 not possible to judge from their paper how firm this exclusion was.…”

Section: Discussionmentioning

confidence: 99%

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Mechanism of chymotrypsin hydrolysis of an ester and an anilide

Stauffer

1971

FEBS Letters

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Section: Discussioncontrasting

confidence: 96%

Section: Resultsmentioning

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Mechanism of chymotrypsin hydrolysis of an ester and an anilide

Stauffer

1971

FEBS Letters

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“…Such an approach makes easy an interpretation of the recent works of Bresler et al [11,12]. They investigated a series of preparations of acyltrypsin with radicals of different sizes (formyl, acetyl, propionyl and trimethylacetyltrypsin).…”

Section: Discussionmentioning

confidence: 99%

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

Березин¹,

Kazanskaya²,

Klyosov³

et al. 1973

European Journal of Biochemistry

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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 ...

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