[Study of penicillin amidase for E. coli. Kinetics of enzymatic hydrolysis of 7-phenylacetamidodeacetoxycephalosporanic acid].

Kinetics of hydrolysis of 7-henylacetamidodeacetoxycephalosporanic acid catalyzed by penicillin amidase as a result of which phenylacetic and 7-aminodeacetoxycephalosporanic acids are formed was studied. The kinetic parameters of the reaction were determined on the basis of both the dependence of the initial rate of enzymatic hydrolysis on the substrate concentration and the analysis of progress kinetic curves of the product accumulation. The values of Km determined by the two methods were equal to (10 +/- 1) muM and kcat (50 +/- 5) sec-1 and (50 +/- 10) sec-1 respectively. The study of the inhibition of the enzymatic hydrolysis by the reaction products showed that phenylacetic and 7-aminodeacetoxycephalosporanic acids were competitive and non-competitive inhibitors of the penicillin amidase activity respectively. The inhibition constants were (55 +/- 8) muM and (12 +/- 1) mM respectively. The physiological role of the enzyme and the effect of the structure of the substrate on the specificity of the enzyme are discussed.