Kinetic analysis of an inhibitor-resistant variant of the OHIO-1 β-lactamase, an SHV-family class A enzyme

The Met(69) --> Ile mutant of the OHIO-1 beta-lactamase, an SHV-family enzyme, is resistant to inactivation by beta-lactamase inhibitors. Analysis of purified Met(69) --> Ile enzyme reveals that its isoelectric point (pI 7.0) and CD spectrum are identical with those of the OHIO-1 enzyme. Levels of beta-lactamase expression in Escherichia coli as determined by immunoblotting are similar for OHIO-1 and Met(69) --> Ile beta-lactamase, The kinetic constants of the Met(69) --> Ile enzyme compared with OHIO-1 are smaller for benzylpenicillin (K-m = 6 mu M compared with 17 mu M; k(cat) = 234 s(-1) compared with 345 s(-1) respectively) and carbenicillin (K-m = 3 mu M compared with 17 mu M; k(cat) = 131 s(-1) compared with 320 s(-1) respectively). For the cephalosporins cephaloridine and 7-(thienyl-2-acetamido)-3-[2-(4-N, N-dimethylaminophenylazo)pyridinium-methyl]-3-cephem-4-carboxylic acid (PADAC), a similar pattern is also seen (K-m = 38 mu M compared with 96 mu M and 6 mu M compared with 75 mu M respectively; k(cat) = 235 s(-1) compared with 1023 s(-1) and 9 s(-1) compared with 50 s(-1) respectively). Consistent with minimum inhibitory concentrations that show resistance to beta-lactam beta-lactamase inhibitors, the apparent K-i values, turnover numbers and partition ratios (k(cat)/k(inact)) for the mechanism-based inactivators clavulanate, sulbactam and tazobactam are increased. The inactivation rate constants (k(inact)) are decreased. The difference in activation energy, a measurement of altered affinity for the wild-type and mutant enzymes leading to acylation of the active site, reveals small energy differences of less than 8.4 kJ/mol. In total, these results suggest that the Met --> Ile substitution at position 69 in the OHIO-1 beta-lactamase alters the active site, primarily affecting the interactions with beta-lactamase inhibitors.