title: Substrate specificity of penicillin amidase from E. coli.
abstract: 1. The kinetic parameters of 12 substrates of penicillin amidase (penicillin amidohydrolase, EC 3.5.1.11) from E. coli have been determined. Most of the penicillin amidase amide substrates containing a phenylacetyl group in the acyl moiety have been shown to have similar catalytic constants of 50 s-1. Substitution of the phenylacetyl group b 2-thienylacetyl group (cephalothin, cephaloridine) having a similar structure leads to a slight decrease in kcat. 2. Nonspecific penicillin amidase substrates, which contain a free amino group in their acyl moiety, are characterized by a strong dependence of kcat, on the structure of the leaving group with Km being constant. To investigate the free amino group influence on the reaction kinetics, pH-dependences of kcat/Km of enzymatic hydrolysis of phenylacetic and D-(-)-alpha-aminophenylacetic acid p-nitroanilides have been studied. It has been shown that enzyme binds the deprotonated form of the substrate only. 3. Under thermodynamically favourable conditions for the synthesis of beta-lactam antibiotics (at low pH), a concentration of the deprotonated substrate form is very low, and the reaction proceeds in the bimolecular regime. The value of the second-order rate constant for the substrate having a free amino group is small even at pH 7.5, and sharply decreases as does the pH. Hence, despite the favourable thermodynamic conditions for the production of all beta-lactam antibiotics, low reaction rate is the basic hindrance for enzymatic synthesis of penicillins and cephalosporins having a free amino group in the acyl moiety.
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism*; D000667:Ampicillin / Q000138:chemical synthesis; D002511:Cephalosporins; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010399:Penicillin Amidase / Q000378:metabolism*; D010406:Penicillins; D013379:Substrate Specificity
chemical_list: D002511:Cephalosporins; D010406:Penicillins; D000667:Ampicillin; D000581:Amidohydrolases; D010399:Penicillin Amidase