The mutation Lys234His yields a class A β-lactamase with a novel pH-dependence

Abstract

The lysine-234 residue is highly conserved in, β-lactamases and in nearly all active-site-serine penicillin-recognizing enzymes. Its replacement by a histidine residue in the Streptomyces albus G class A β-lactamase yielded an enzyme the pH-dependence of which was characterized by the appearance of a novel pK, which could be attributed to the newly introduced residue. At low pH, the k(cat) value for benzylpenicillin was as high as 50 % of that of the wild-type enzyme, demonstrating that an efficient active site was maintained. Both k(cat) and k(cat)/K(m) dramatically decreased above pH 6 but the decrease in k(cat)/K(m) could not be attributed to larger K(m) values. Thus a positive charge on the side chain of residue 234 appears to be more essential for transition-state stabilization than for initial recognition of the substrate ground state.