Kinetic Comparison of Penicillin Amidase Catalysed Transfer of Nonspecific and Specific Acyl Moieties to 7-Aminodeacetoxycephalosporanic Acid

Kinetic evidence for formation of an acylenzyme-nucleophile complex is presented for the penicillin amidase (EC 3.5.1.11) catalysed transfer of nonspecific (2-benzoxazolon-3-yl-acetyl) and specific (phenylacetyl) acyl moieties to 7-aminodeacetoxycephalosporanic acid. The specific and nonspecific acylenzymes differ in their binding constants for 7-aminodeacetoxy-cephalosporanic acid, in their values of transferase to hydrolase ratio, "specificity constants" and maximal yields in kinetically controlled cephem synthesis. The 2-benzoxazolon-3-yl-acetyl-penicillin amidase has a higher affinity for the nucleophile (KN=1.4mM) and the acylen-zyme-nucleophile complex formed is quantitatively converted into cephem, the deacylation by water being negligible. The value of transferase to hydrolase ratio is an order of magnitude higher compared to that for the specific acyl donor, while the "specificity constant" for the respective cephem synthesis is two orders of magnitude lower. The phenylacetyl-penicillin amidase has a lower affinity for 7-aminodeacetoxycephalosporanic acid (KN = 8.4 mM), the acylenzyme-nucleophile complex formed undergoing both transfer and hydrolysis. The observed differences imply that the nonspecific acyl donor binds in a different way which enhances the nucleophile specificity. This results in a higher yield in the kinetically controlled synthesis of the respective cephem.