Penicillin acylases as amidohydrolases and acyl transfer catalysts

Abstract

The hypothesis that an acyl-enzyme intermediate of the esterases from Xanthomonas cirri and Acetobacter turbidans is responsible for the acylation of 7-aminodeacetoxycephalosporanic acid and similar compounds by esters of amino acids, recently proposed by Kate, also accounts for the kinetics of hydrolysis of penicillin G by the amidohydrolase from E. coli and other reactions of the enzyme. Some further implications of the mechanism are derived and discussed. In recent studies concerning the acylation of 7-aminodeacetoxycephalosporanic acid (7-ADCA) by the methyl ester of D-phenyl glycine and of similar acylations catalyzed by the esterases from x. cirri and A. turbidans (Kato and coworkers 1980a, 1980b; Kate, 1980) it has been deduced that the reactions proceed via an acy!-enzyme intermediate. Kinetics of the reactions indicate a rate determining acylation of the enzyme by the ester, followed by a fast transfer of the acyl radical to the amino group of 7-ADCA or water, which compete for the acyl group as accepters. The purpose of this communication is to show that this mechanism has broader implications. In particular it accounts for * qhis paper is dedicated to Prof. G. Manecke, a pioneer of immobilized enzyme technology, with oongratulations to his 65thbirthday. 107

Structure, Processing and Catalytic Action of Penicillin Acylase

“…It was noted by Konecny (1981) that the ability of penicillin acylase to catalyse acyl group transfers which include trans-acylation could be explained readily by the formation of an acyl enzyme intermediate. Phenylacetic acid would be predicted to be a competitive inhibitor and, assuming direct attack of the nucleophfle on the acyl enzyme, 6-APA would be a non-competitive inhibitor.…”

Chemical modification of serine at the active site of penicillin acylase from Kluyvera citrophila

“…In summary, the simplest interpretation of the present results is that Ser-290 is the site modified by reaction with PMSF. The kinetics of transacylation reactions catalysed by penicillin acylase (Konecny, 1981) support an acyl-enzyme mechanism, and we suggest Ser-290 as a candidate site for this acylation.…”

Biotransformations Catalyzed by Multimeric Enzymes: Stabilization of Tetrameric Ampicillin Acylase Permits the Optimization of Ampicillin Synthesis under Dissociation Conditions

“…Therefore the ampicillin synthetic yields are very poor even using high excesses of acyl donors, usually under 50%. This has promoted an intense evaluation of many other enzyme sources − including the one reported in this paper. However, the results have not been fully satisfactory, in many cases because of the limits established by the enzyme stability.…”