PH-DEPENDENCE OF AND KINETIC SOLVENT ISOTOPE EFFECTS ON THE METHANOLYSIS AND HYDROLYSIS OF BETA-LACTAMS CATALYZED BY CLASS-C BETA-LACTAMASE

The methanolysis of benzylpenicillin is catalyzed by Enterobacter cloa cae P99 class C beta-lactamase and the pH dependence of k(cat) indicat es that the catalytic groups involved of pK(a) ca. 5 and 10 are the sa me as those for hydrolysis. The kinetic solvent isotope effect (KSIE) k(cat)(H)2(O)/k(cat)(D)2(O) is 1.42 for both the hydrolysis and methan olysis of benzylpenicillin. However, there is an inverse KSIE on k(cat )/k(m) of 0.83 +/- 0.05 for the hydrolysis of benzylpenicillin and cep haloridine. There is also an abnormally high shift in the low pK, on g oing from H2O to D2O of 0.85 +/- 0.15 from the pH dependence of both k (cat) and k(cat)/K-m for both methanolysis and hydrolysis. The D2O shi ft on the high pK(a) of ca. 10 is the normal value of ca. 0.4. These r esults are consistent with a strongly hydrogen bonded system acting as the general base catalyst and low fractionation factors for the hydro gens involved. It is probable that this represents the tyrosine phenox ide ion hydrogen bonded to two lysine ammonium ions.