Kinetic analysis of an inhibitor-resistant variant of the OHIO-1 beta-lactamase, an SHV-family class A enzyme.
The Met69-->Ile mutant of the OHIO-1 beta-lactamase, an SHV-family enzyme, is resistant to inactivation by beta-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 beta-lactamase expression in Escherichia coli as determined by immunoblotting are similar for OHIO-1 and Met69-->Ile beta-lactamase. The kinetic constants of the Met69-->Ile enzyme compared with OHIO-1 are smaller for benzylpenicillin (Km = 6 microM compared with 17 microM; kcat = 234 s-1 compared with 345 s-1 respectively) and carbenicillin (Km = 3 microM compared with 17 microM; 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 microM compared with 96 microM and 6 microM compared with 75 microM 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 beta-lactam beta-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 beta-lactamase alters the active site, primarily affecting the interactions with beta-lactamase inhibitors.
Lin
S
S
Research Service, Department of Veterans Affairs Medical Center, 10701 East Boulevard, Cleveland, OH 44105, USA.
Thomas
M
M
Shlaes
D M
DM
Rudin
S D
SD
Knox
J R
JR
Anderson
V
V
Bonomo
R A
RA
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002942:Circular Dichroism; D019818:Clavulanic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008715:Methionine / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D010397:Penicillanic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D017354:Point Mutation; D011487:Protein Conformation; D013407:Sulbactam / Q000494:pharmacology; D000078142:Tazobactam; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D007532:Isoleucine; D019818:Clavulanic Acid; D010397:Penicillanic Acid; D008715:Methionine; D001618:beta-Lactamases; D013407:Sulbactam; D000078142:Tazobactam