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

The Met⁶⁹ → Ile mutant of the OHIO-1 β-lactamase, an SHV-family enzyme, is resistant to inactivation by β-lactamase inhibitors. Analysis of purified Met⁶⁹ → Ile enzyme reveals that its isoelectric point (pI 7.0) and CD spectrum are identical with those of the OHIO-1 enzyme. Levels of β-lactamase expression in Escherichia coli as determined by immunoblotting are similar for OHIO-1 and Met⁶⁹ → Ile β-lactamase. The kinetic constants of the Met⁶⁹ → Ile enzyme compared with OHIO-1 are smaller for benzylpenicillin (K_m = 6 µM compared with 17 µM; k_cat = 234 s^-1 compared with 345 s^-1 respectively) and carbenicillin (K_m = 3 µM compared with 17 µM; 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 µM compared with 96 µM and 6 µM compared with 75 µM 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 β-lactam β-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 β-lactamase alters the active site, primarily affecting the interactions with β-lactamase inhibitors.