Amino-enzyme intermediates in pepsin-catalyzed reactions

Silver, Marc S.; Stoddard, M. Palmer

doi:10.1021/bi00752a008

Cited by 20 publications

(16 citation statements)

References 21 publications

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“…The high yield of transfer when substrates at millimolar concentrations act as acceptors suggests that a high degree of binding specificity is required for a compound to act as acceptor. Silver & Stoddard (1972) questioned the relevance of the amino intermediate in pepsin-catalysed transpeptidation reactions to hydrolytic reactions. One of the arguments used was that the pH-dependence of transpeptidation does not follow that of hydrolysis and that at low pH no transpeptidation occurs.…”

Section: Vol 147mentioning

confidence: 99%

Acyl intermediates in penicillopepsin-catalysed reactions and a discussion of the mechanism of action of pepsins.

Takahashi

Hofmann

1975

Biochemical Journal

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Penicillopepsin catalyses transpeptidation reactions involving the transfer of the N-terminal amino acids of suitable substrates via covalent acyl intermediates to acceptor peptides, usually the substrate. The major products obtained when Phe-Tyr-Thr-Pro-Lys-Ala and Met-Leu-Gly were used as substrates were Phe-Phe and Met-Met respectively. With Met-Leu-Gly the tetrapeptide Met-Met-Leu-Gly was observed as probable intermediate. Co-incubation of Leu-Tyr-Leu and Phe-Tyr-Thr-Pro-Lys-Ala led to the formation of Leu-Phe and Phe-Leu as well as Leu-Leu and Phe-Phe. No reaction was observed with tripeptides in which the first or second amino acid is glycine. It appears that two amino aicds with large hydrophobic residues are needed for the transpeptidation reaction. Nucleophilic compounds other than peptides, such as hydroxylamine, aliphatic alcohols and dinitrophenylhydrazine, were not acceptors for the acyl group. Leucine, phenylalanine and leucine methyl ester also had no effect on the reaction. The transpeptidation reaction proceeded readily at pH 3.6 and 4.7. At pH 6.0 the reaction was slow and at pH 1.9 little or no transpeptidation was observed. Porcine pepsin catalyses similar transpeptidation reactions. Sequence studies show that porcine pepsin and penicillopepsin are homologous. The present study also suggests that they have a very similar mechanism. Evidence available at this time indicates that the mechanism of these enzymes is complex and may be modulated by secondary substrate-enzyme interactions. A hypothesis is presented which proposes that pepsin-catalysed reactions proceed via different covalent intermediates (amino-intermediates or acylintermediates) depending on the nature of the substrate. The possibility that some reactions do not involve covalent intermediates is also discussed.

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Section: Vol 147mentioning

confidence: 99%

Acyl intermediates in penicillopepsin-catalysed reactions and a discussion of the mechanism of action of pepsins.

Takahashi

Hofmann

1975

Biochemical Journal

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“…Consequently, stopped-flow measurements of the rate of decrease of the fluorescence of the en- (14), some findings (15)(16)(17) are inconsistent with this conclusion, and further work is needed. The present studies were performed in the hope of contributing to the solution of this problem, through a comparison of the values of k2 and K, estimated from stopped-flow fluorescence measurements with the values of kcat and Km obtained under steady-state conditions, where the rate of release of the Phe-OP4P product was measured by means of its reaction with fluorescamine (5).…”

mentioning

confidence: 99%

Kinetics of action of pepsin on fluorescent peptide substrates.

Sachdev¹,

Fruton²

1975

Proc. Natl. Acad. Sci. U.S.A.

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Oligopeptide substrates of porcine pepsin (E) of the type A-Phe-Phe-B (S) that are cleaved solely at the PhePhe bond under the conditions of these studies, and bearing an amino-terminal fluorescent probe group (mansyl or dansyl), have been used for stopped-flow measurements of the rate of formation of the A-Phe product. These experiments were conducted under conditions of [El >> [S], and the kinetic data were compared with those obtained under conditions of [S] >> [El for the formation of the Phe-B product (the same in all cases). The results for substrates with A = mansyl-Gly, mansyl-Gly-Gly, and dansyl-Gly-Gly support the conclusion that the rate-limiting step in the over-all catalytic process is associated with the scission of the Phe-Phe bond in the first detectable ES complex. Although the rate of this step varies widely with the nature of the A portion of A-Phe-Phe-B, the magnitude of the dissociation constant of ES is relatively invariant. This supports the view that, in the cleavage of oligopeptide substrates by pepsin, secondary enzyme-substrate interactions may cause conformational changes at the catalytic site, and that a portion of the total binding energy may be used for the attainment of the transition state in the bondbreaking step. With substrates that are hydrolyzed extremely rapidly (A = dansyl-Gly-Ala, dansyl-Ala-Ala), the rate of formation of the A-Phe product appears to be faster than the steady-state rate, suggesting that an additional step has become kinetically significant in the over-all process. This step may be associated with the return of the conformation of the active site to its original state. In previous studies from this laboratory (1-6), a series of synthetic oligopeptide substrates of the type A-Phe-Phe-OP4P was described for porcine pepsin and other acid proteinases. The pyridylpropyloxy (OP4P) (8) were consistent with the conclusion that the values of Km estimated from steady-state kinetic measurements approximate the value of K,, the dissociation constant of the initial enzyme-substrate complex, and that the rate-limiting step in the over-all catalytic process occurs during the transformation of this complex. In the experiments described below, the validity of this conclusion was tested by means of stopped-flow fluorescence measurements of the kinetics of the cleavage of substrates of the type A-Phe-Phe-OP4P by porcine pepsin and Rhizopus-pepsin (9) has been applied to the study of the mechanism of the action of papain (11), chymotrypsin (10, 12), and trypsin (13).In the present work, advantage was taken of the earlier finding (3, 4, 6) that the fluorescence of the dansyl (Dns) or mansyl (Mns) group of compounds such as Dns-(or Mns-) Gly-Gly-Phe-Phe-OP4P is greatly enhanced when they are bound by pepsin and that cleavage products (e.g., Dns-GlyGly-Phe) bearing the fluorescent probe group are bound much more weakly. Consequently, stopped-flow measurements of the rate of decrease of the fluorescence of the en- MATERIALS AND METHODSPorcine pepsin was prepared f...

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“…Numerous reports (94)(95)(96)(97) have confirmed and extended the finding that acyl dipeptides, such as Z-Phe-Tyr, readily undergo such transpeptidation reactions, with the formation (in this case) of detectable amounts of Tyr-Tyr (arising from the cleavage of the transpeptidation product ZPhe-Tyr-Tyr). In particular, Antonov et al (97) have used a spectrophotometric method for following pepsin-catalyzed transpeptidation with Z-Phe(4NO.J as the acceptor molecule.…”

Section: Rco-nhr" + E E E[rco-nhr'] E E-nhr' + Rcooh R"co0h + E-nhr' mentioning

confidence: 81%

“…An estimate has been made of the relative rates of the hydrolysis of Ac-Phe-Tyr (or Ac-Tyr-Tyr) and of the formation of Z-Phe(4NOz)-Tyr from these substrates. It is noteworthy that these rates With some esters or amides of Ac-Phe-Phe, however, no transpeptidation (with 'H-labeled Ac-Phe as the acceptor) could be found (96). Antonov et al (97) have suggested that in this case the initial rate of hydrolysis is so much greater than the rate of transpeptidation that no transfer product is detectable, and it has been reported (97a) that the ymorpholinopropyl amide of Ac-Phe-Phe undergoes a transpeptidation reaction with Z-Phe(4NOz) as the acceptor.…”

Section: Rco-nhr" + E E E[rco-nhr'] E E-nhr' + Rcooh R"co0h + E-nhr' mentioning

confidence: 85%