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Streptomyces K15 active-site serine DD-transpeptidase: specificity profile for peptide, thiol ester and ester carbonyl donors and pathways of the transfer reactions.

ABSTRACT: The Streptomyces K15 transferase is a penicillin-binding protein presumed to be involved in bacterial wall peptidoglycan crosslinking. It catalyses cleavage of the peptide, thiol ester or ester bond of carbonyl donors Z-R1-CONH-CHR2-COX-CHR3-COO- (where X is NH, S or O) and transfers the electrophilic group Z-R1-CONH-CHR2-CO to amino acceptors via an acyl-enzyme intermediate. Kinetic data suggest that the amino acceptor behaves as a simple alternative nucleophile at the level of the acyl-enzyme in the case of thiol ester and ester donors, and that it binds to the enzyme.carbonyl donor Michaelis complex and influences the rate of enzyme acylation by the carbonyl donor in the case of amide donors. Depending on the nature of the scissile bond, the enzyme has different requirements for substituents at positions R1, R2 and R3.

SUBMITTER: Grandchamps J

PROVIDER: S-EPMC1136653 | biostudies-other | 1995 Apr

REPOSITORIES: biostudies-other

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Project description:Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic-enzyme complexes. At saturation, the molar ratio of chromogenic cephalosporin 87-312 to enzyme was 1.3:1, but this discrepancy might be due to a lack of accuracy in the measurement of the antibiotic. Spectrophotometric studies showed that binding of cephaloridine and cephalosporin 87-312 to the enzyme caused opening of their beta-lactam rings. Benzylpenicillin and cephalosporin 87-312 competed for the same site on the free enzyme, suggesting that binding of benzylpenicillin also resulted in the opening of its beta-lactam ring. In Tris-NaCl-MgCl(2) buffer at pH7.7 and 37 degrees C, the rate constants for the dissociation of the antibiotic-enzyme complexes were 2.8x10(-6), 1.5x10(-6) and 0.63x10(-6)s(-1) (half-lives 70, 130 and 300h) for benzylpenicillin, cephalosporin 87-312 and cephaloridine respectively. During the process, the protein underwent reactivation. The enzyme that was regenerated from its complex with benzylpenicillin was as sensitive to fresh benzylpenicillin as the native enzyme. With [(14)C]benzylpenicillin, the released radioactive compound was neither benzylpenicillin nor benzylpenicilloic acid. The Streptomyces R39 enzyme thus behaved as a beta-lactam-antibiotic-destroying enzyme but did not function as a beta-lactamase. Incubation at 37 degrees C in 0.01m-phosphate buffer, pH7.0, and in the same buffer supplemented with sodium dodecyl sulphate caused a more rapid reversion of the [(14)C]benzylpenicillin-enzyme complex. The rate constants were 1.6x10(-5)s(-1) and 0.8x10(-4)s(-1) respectively. Under these conditions, however, there was no concomitant reactivation of the enzyme and the released radioactive compound(s) appeared not to be the same as before. The Streptomyces R39 enzyme and the exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R61 appeared to differ from each other with regard to the topography of their penicillin-binding site.

Dataset Information

Streptomyces K15 active-site serine DD-transpeptidase: specificity profile for peptide, thiol ester and ester carbonyl donors and pathways of the transfer reactions.

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