Reference : Binding of beta-lactam antibiotics to the exocellular DD-carboxypeptidase-transpeptid...
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
Life sciences : Microbiology
http://hdl.handle.net/2268/83095
Binding of beta-lactam antibiotics to the exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R39
English
Frère, Jean-Marie mailto [Université de Liège - ULg > Faculté de Médecine, Institut de Botanique > Service de Microbiologie > >]
Ghuysen, Jean-Marie [Université de Liège - ULg > Faculté de Médecine, Institut de Botanique > Service de Microbiologie > > >]
Reynolds, Peter E. [University of Cambridge > Department of Biochemistry > Sub-Department of Chemical Microbiology > >]
Moreno, Ramon [Universidad de los Andes > Facultad de Ciencias > > >]
1974
Biochemical Journal
Portland Press
143
1
241-249
Yes (verified by ORBi)
International
0264-6021
1470-8728
London
United Kingdom
[en] binding, competitive ; carboxypeptidases/*metabolism ; cephaloridine/*metabolism ; cephalosporins/metabolism ; chromatography, Thin Layer ; circular dichroism ; dithionitrobenzoic acid ; electrophoresis, paper ; hydrolysis ; iodoacetates ; kinetics ; molecular weight ; muramoylpentapeptide carboxypeptidase/*metabolism ; penicillin G/*metabolism ; penicillinase ; protein binding ; scintillation counting ; sodium dodecyl sulfate ; spectrometry, fluorescence ; streptomyces/*enzymology
[en] 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.
Researchers ; Professionals
http://hdl.handle.net/2268/83095

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