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 cloacae P99 class C P-lactamase and the pH dependence of kcat indicates that the catalytic groups involved of PKa ca. 5 and 10 are the same as those for hydrolysis. The kinetic solvent isotope effect (KSIE) e:/e$) is 1.42 for both the hydrolysis and methanolysis of benzylpenicillin. However, there is an inverse KSIE on kJKm of 0.83 f 0.05 for the hydrolysis of benzylpenicillin and cephaloridine. There is also an abnormally high shift in the low PKa on going from H20 to D20 of 0.85 f 0.15 from the pH dependence of both kcat and &/Km for both methanolysis and hydrolysis. The D20 shift on the high PKa 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. The major cause of the resistance of some bacteria to the normally lethal action of /3-lactam antibiotics is the ability of the bacteria to produce P-lactamase enzymes, which catalyze the hydrolysis of the j3-lactam of penicillins 1 (Scheme 1) and cephalosporins. The most prevalent and clinically important P-lactamases are the class A and C type which are both serine enzymes and act by a mechanism involving the formation of a relatively unstable acyl enzyme (EA) intermediate, eq 1.' Although there have been several crystal structures of P-lactamases and their derivatives rep~rted~~~,~ and the nature and degree of the conservation of the amino acid residues in and near the active site are known,5 there is little detailed evidence of the groups presumed to be involved with the necessary proton tfansfer Comparison with serine proteases would indicate the need for general acid-base catalysis.8 The main contenders for these roles are, in class A