pH Dependence of and Kinetic Solvent Isotope Effects on the Methanolysis and Hydrolysis of β-Lactams Catalyzed by Class C β-Lactamase

The methanolysis of benzylpenicillin is catalyzed by Enterobacter cloacae P99 class C β-lactamase and the pH dependence of k_cat indicates that the catalytic groups involved of pK_a ca. 5 and 10 are the same as those for hydrolysis. The kinetic solvent isotope effect (KSIE) is 1.42 for both the hydrolysis and methanolysis of benzylpenicillin. However, there is an inverse KSIE on of 0.83 ± 0.05 for the hydrolysis of benzylpenicillin and cephaloridine. There is also an abnormally high shift in the low pK_a on going 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 shift on the high pK_a of ca. 10 is the normal value of ca. 0.4. These results are consistent with a strongly hydrogen bonded system acting as the general base catalyst and low fractionation factors for the hydrogens involved. It is probable that this represents the tyrosine phenoxide ion hydrogen bonded to two lysine ammonium ions.