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 (Km = 6 µM compared with 17 µM; kcat = 234 s-1 compared with 345 s-1 respectively) and carbenicillin (Km = 3 µM compared with 17 µM; kcat = 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 (Km = 38 µM compared with 96 µM and 6 µM compared with 75 µM respectively; kcat = 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 Ki values, turnover numbers and partition ratios (kcat/kinact) for the mechanism-based inactivators clavulanate, sulbactam and tazobactam are increased. The inactivation rate constants (kinact) 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.
Kinetic analysis of an inhibitor-resistant variant of the OHIO-1 β-lactamase, an SHV-family class A enzyme
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Shan LIN, Mary THOMAS, David M. SHLAES, Susan D. RUDIN, James R. KNOX, Vernon ANDERSON, Robert A. BONOMO; Kinetic analysis of an inhibitor-resistant variant of the OHIO-1 β-lactamase, an SHV-family class A enzyme. Biochem J 15 July 1998; 333 (2): 395–400. doi: https://doi.org/10.1042/bj3330395
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