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Effects of nucleophiles on the breakdown of the benzylpenicilloyl-enzyme complex EI formed between benzylpenicillin and the exocellular DD-carboxypeptidase--transpeptiase of Streptomyces strain R61.

ABSTRACT: Serine is one of the enzyme residues with which benzylpenicillin collides as a result of its binding to the Streptomyces strain-R61 DD-carboxypeptidase-transpeptidase enzyme. Nucleophilic attack occurs on C(7) of the bound antibiotic molecule with formation of a benzylpenicilloyl-serine ester linkage, i.e. formation of the benzylpenicilloyl-enzyme EI complex. To reject the bound penicilloyl moiety and consequently to recover its initial activities, the strain-R61 enzyme has developed two possible mechanisms. Pathway A is a direct attack of the serine ester linkage by an exogenous nucleophile, resulting in the transfer of the benzylpenicilloyl moiety to this nucleophile. In pathway B, the benzylpenicilloyl moiety is first fragmented by C(5)-C(6) cleavage and the enzyme-bound phenylacetylglycyl residue thus produced is in turn transferred to the nucleophile. Pathway B occurs with water, glycylglycine and other amino compounds. Both pathways A and B occur with glycerol, other ROH nucleophiles and neutral hydroxylamine. The nucleophilic attacks are enzyme-catalysed.

SUBMITTER: Marquet A

PROVIDER: S-EPMC1186457 | biostudies-other | 1979 Mar

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.

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Effects of nucleophiles on the breakdown of the benzylpenicilloyl-enzyme complex EI formed between benzylpenicillin and the exocellular DD-carboxypeptidase--transpeptiase of Streptomyces strain R61.

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