title: Kinetic studies on the mechanism of the penicillin amidase-catalysed synthesis of ampicillin and benzylpenicillin.
abstract: Hydrophobic protein chromatography was used to prepare homogeneous fractions of penicillin amidase (EC 3.5.1.11) from E. coli. The apparent ratios of the rate constants for the deacylation of the acyl-penicillin amidase formed in the hydrolysis of phenylacetylglycine or D-phenylglycine methyl ester, by H2O and 6-aminopenicillanic acid (6-APA), were determined at different concentrations of the latter compound. The ratios were obtained from direct measurements of the initial rates of formation of phenylacetic acid and benzylpenicillin or D-phenylglycine and ampicillin. For the semisynthesis of ampicillin as well as of benzylpenicillin the ratio was found to depend on the concentration of 6-APA. This was observed for heterogeneous and homogeneous enzyme preparations. These results show that 6-APA must be bound to the acyl-enzyme before the deacylation, yielding ampicillin and benzylpenicillin, occurs. The dissociation constant KN for the formation of the complex was estimated to be approximately 10mM. This mechanism in which acyl-enzyme with and without bound nucleophile is involved, is in agreement with the principle of microscopic reversibility. Both acyl-enzymes can be deacylated by H2O. The finding that there is a specific binding site for 6-APA adjacent to the binding site for the phenylacetyl-(D-phenylglycyl-) group in the active site of the enzyme is supported by the observation that 6-APA acts as a mixed inhibitor in the hydrolysis of D-phenylglycine methyl ester. The ionic strength dependence indicates that the binding site for 6-APA of the acyl-enzyme is positively charged.
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism*; D000667:Ampicillin / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology; D007525:Isoelectric Focusing; D007700:Kinetics; D009994:Osmolar Concentration; D010397:Penicillanic Acid / Q000378:metabolism / Q000031:analogs & derivatives; D010399:Penicillin Amidase / Q000378:metabolism*; D010400:Penicillin G / Q000096:biosynthesis*
chemical_list: D000667:Ampicillin; D010397:Penicillanic Acid; D000581:Amidohydrolases; D010399:Penicillin Amidase; D010400:Penicillin G; C004408:aminopenicillanic acid