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.
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Research Article|
July 15 1998
Kinetic analysis of an inhibitor-resistant variant of the OHIO-1 β-lactamase, an SHV-family class A enzyme
Shan LIN;
Shan LIN
*Research Service, Department of Veterans Affairs Medical Center, 10701 East Boulevard, Cleveland, OH 44105, U.S.A.
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Mary THOMAS;
Mary THOMAS
*Research Service, Department of Veterans Affairs Medical Center, 10701 East Boulevard, Cleveland, OH 44105, U.S.A.
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David M. SHLAES;
David M. SHLAES
*Research Service, Department of Veterans Affairs Medical Center, 10701 East Boulevard, Cleveland, OH 44105, U.S.A.
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Susan D. RUDIN;
Susan D. RUDIN
*Research Service, Department of Veterans Affairs Medical Center, 10701 East Boulevard, Cleveland, OH 44105, U.S.A.
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James R. KNOX;
James R. KNOX
†Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269-3125, U.S.A.
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Vernon ANDERSON;
Vernon ANDERSON
‡Department of Biochemistry, Case Western Reserve University, School of Medicine, 2109 Adelbert Road, Cleveland, OH 44105, U.S.A.
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Robert A. BONOMO
Robert A. BONOMO
1
*Research Service, Department of Veterans Affairs Medical Center, 10701 East Boulevard, Cleveland, OH 44105, U.S.A.
§Geriatric CARE Center, University Hospitals of Cleveland, 12200 Fairhill Road, Cleveland, OH 44120, U.S.A.
1To whom correspondence should be addressed at Geriatric CARE Center (e-mail rab14@po.cwru.edu).
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Publisher: Portland Press Ltd
Received:
November 14 1997
Revision Received:
March 26 1998
Accepted:
April 08 1998
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 333 (2): 395–400.
Article history
Received:
November 14 1997
Revision Received:
March 26 1998
Accepted:
April 08 1998
Citation
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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