Deacylation of penicilloylated penicillin-binding proteins from Micrococcus luteus: kinetic study of thiol-promoted release of the bound penicilloyl moiety.
The stability of covalent complexes obtained by labelling penicillin-binding proteins 1-6 from Micrococcus luteus with a radioactive derivative of ampicillin has been examined in the presence of thiols. When the incubation medium contained only 1 mM 2-mercaptoethanol, the complexes were almost unaffected for at least 1 h. If 20 mM dithiothreitol was also included in the medium, the amount of bound radioactivity decreased throughout the incubation period. The breakdown of the complexes derived from penicillin-binding proteins 4 and 5 proceeded very slowly, following an apparent first-order kinetics, whereas the kinetics of deacylation of other penicillin-binding proteins exhibited a biphasic pattern with an initial fast phase followed by a slow one, each of which could be approximated by an apparent first-order reaction. This behavior is explained adequately by a two-step mechanism: the penicilloylated penicillin-binding proteins are first deacylated in a reversible exchange with the added thiol, giving rise to an intermediate thioester; once formed, this intermediate is hydrolysed irreversibly. A simple graphical method has been devised to deduce rate constants from the time course of the reaction. Theoretical curves have been constructed, and they fit experimental data satisfactorily. The results point out that added thiols may effectively interfere with the quantitation of penicillin-binding proteins; therefore, the stability of penicilloylated penicillin-binding proteins should be checked carefully when these protecting agents are included in membrane extracts or incubation media.
D000667:Ampicillin / Q000031:analogs & derivatives / Q000032:analysis; D001426:Bacterial Proteins / Q000032:analysis*; D001665:Binding Sites; D002352:Carrier Proteins / Q000032:analysis*; D006602:Hexosyltransferases; D007700:Kinetics; D008433:Mathematics; D008837:Micrococcus; D008956:Models, Chemical; D002267:Muramoylpentapeptide Carboxypeptidase / Q000032:analysis*; D010397:Penicillanic Acid / Q000031:analogs & derivatives; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D013438:Sulfhydryl Compounds
D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D013438:Sulfhydryl Compounds; C008782:penicilloic acid; C050124:N-propionylampicillin; D000667:Ampicillin; D010397:Penicillanic Acid; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase