Effects of sulfhydryl reagents on the binding and release of penicillin G by D-alanine carboxypeptidase IA of Escherichia coli.
Purified D-alanine carboxypeptidase IA of Escherichia coli is inhibited by penicillin G and binds penicillin G reversibly. The binding of penicillin to the enzyme is relatively insensitive to sulfhydryl reagents, while release of penicillin from the enzyme is severely inhibited by these reagents. The inhibition of release parallels the inhibition of carboxypeptidase activity by the sulfhydryl reagents. In the presence of the sulfhydryl reagent p-chloromercuribenzoate, an acyl-enzyme intermediate, produced by the reaction of carboxypeptidase IA with diacetyl-L-lysyl-D-alanyl-D-alanine, accumulates and can be isolated. These results indicate that binding of penicillin to carboxypeptidase IA occurs by an acylation step of the carboxypeptidase reaction, while penicillin release occurs by a deacylation step of the reaction. Only the latter is inhibited by sulfhydryl reagents.
D000215:Acylation; D002268:Carboxypeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006898:Hydroxylamines / Q000494:pharmacology; D008623:Mercaptoethanol / Q000494:pharmacology; D002267:Muramoylpentapeptide Carboxypeptidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010400:Penicillin G / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D006898:Hydroxylamines; D013439:Sulfhydryl Reagents; D008623:Mercaptoethanol; D002268:Carboxypeptidases; D002267:Muramoylpentapeptide Carboxypeptidase; D010400:Penicillin G