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. 1998 Jul 15;333(Pt 2):395–400. doi: 10.1042/bj3330395

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

S Lin 1, M Thomas 1, D M Shlaes 1, S D Rudin 1, J R Knox 1, V Anderson 1, R A Bonomo 1
PMCID: PMC1219605  PMID: 9735103

Abstract

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.

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Selected References

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  1. Ambler R. P., Coulson A. F., Frère J. M., Ghuysen J. M., Joris B., Forsman M., Levesque R. C., Tiraby G., Waley S. G. A standard numbering scheme for the class A beta-lactamases. Biochem J. 1991 May 15;276(Pt 1):269–270. doi: 10.1042/bj2760269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ambler R. P. The structure of beta-lactamases. Philos Trans R Soc Lond B Biol Sci. 1980 May 16;289(1036):321–331. doi: 10.1098/rstb.1980.0049. [DOI] [PubMed] [Google Scholar]
  3. Bonomo R. A., Currie-McCumber C., Shlaes D. M. OHIO-1 beta-lactamase resistant to mechanism-based inactivators. FEMS Microbiol Lett. 1992 Apr 1;71(1):79–82. doi: 10.1016/0378-1097(92)90545-y. [DOI] [PubMed] [Google Scholar]
  4. Bonomo R. A., Dawes C. G., Knox J. R., Shlaes D. M. Complementary roles of mutations at positions 69 and 242 in a class A beta-lactamase. Biochim Biophys Acta. 1995 Feb 22;1247(1):113–120. doi: 10.1016/0167-4838(94)00187-l. [DOI] [PubMed] [Google Scholar]
  5. Bonomo R. A., Dawes C. G., Knox J. R., Shlaes D. M. beta-Lactamase mutations far from the active site influence inhibitor binding. Biochim Biophys Acta. 1995 Feb 22;1247(1):121–125. doi: 10.1016/0167-4838(94)00188-m. [DOI] [PubMed] [Google Scholar]
  6. Bush K., Jacoby G. A., Medeiros A. A. A functional classification scheme for beta-lactamases and its correlation with molecular structure. Antimicrob Agents Chemother. 1995 Jun;39(6):1211–1233. doi: 10.1128/aac.39.6.1211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bush K., Macalintal C., Rasmussen B. A., Lee V. J., Yang Y. Kinetic interactions of tazobactam with beta-lactamases from all major structural classes. Antimicrob Agents Chemother. 1993 Apr;37(4):851–858. doi: 10.1128/aac.37.4.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chaibi E. B., Péduzzi J., Farzaneh S., Barthélémy M., Sirot D., Labia R. Clinical inhibitor-resistant mutants of the beta-lactamase TEM-1 at amino-acid position 69. Kinetic analysis and molecular modelling. Biochim Biophys Acta. 1998 Jan 15;1382(1):38–46. doi: 10.1016/s0167-4838(97)00127-1. [DOI] [PubMed] [Google Scholar]
  9. Delaire M., Labia R., Samama J. P., Masson J. M. Site-directed mutagenesis at the active site of Escherichia coli TEM-1 beta-lactamase. Suicide inhibitor-resistant mutants reveal the role of arginine 244 and methionine 69 in catalysis. J Biol Chem. 1992 Oct 15;267(29):20600–20606. [PubMed] [Google Scholar]
  10. Edelhoch H. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry. 1967 Jul;6(7):1948–1954. doi: 10.1021/bi00859a010. [DOI] [PubMed] [Google Scholar]
  11. Farzaneh S., Chaibi E. B., Peduzzi J., Barthelemy M., Labia R., Blazquez J., Baquero F. Implication of Ile-69 and Thr-182 residues in kinetic characteristics of IRT-3 (TEM-32) beta-lactamase. Antimicrob Agents Chemother. 1996 Oct;40(10):2434–2436. doi: 10.1128/aac.40.10.2434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Giakkoupi P., Tzelepi E., Legakis N. J., Tzouvelekis L. S. Substitution of Arg-244 by Cys or Ser in SHV-1 and SHV-5 beta-lactamases confers resistance to mechanism-based inhibitors and reduces catalytic efficiency of the enzymes. FEMS Microbiol Lett. 1998 Mar 1;160(1):49–54. doi: 10.1111/j.1574-6968.1998.tb12889.x. [DOI] [PubMed] [Google Scholar]
  13. Imtiaz U., Billings E. M., Knox J. R., Mobashery S. A structure-based analysis of the inhibition of class A beta-lactamases by sulbactam. Biochemistry. 1994 May 17;33(19):5728–5738. doi: 10.1021/bi00185a009. [DOI] [PubMed] [Google Scholar]
  14. Johnson B. H., Hecht M. H. Recombinant proteins can be isolated from E. coli cells by repeated cycles of freezing and thawing. Biotechnology (N Y) 1994 Dec;12(13):1357–1360. doi: 10.1038/nbt1294-1357. [DOI] [PubMed] [Google Scholar]
  15. Joris B., Ghuysen J. M., Dive G., Renard A., Dideberg O., Charlier P., Frère J. M., Kelly J. A., Boyington J. C., Moews P. C. The active-site-serine penicillin-recognizing enzymes as members of the Streptomyces R61 DD-peptidase family. Biochem J. 1988 Mar 1;250(2):313–324. doi: 10.1042/bj2500313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Knox J. R. Extended-spectrum and inhibitor-resistant TEM-type beta-lactamases: mutations, specificity, and three-dimensional structure. Antimicrob Agents Chemother. 1995 Dec;39(12):2593–2601. doi: 10.1128/aac.39.12.2593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Medeiros A. A. Evolution and dissemination of beta-lactamases accelerated by generations of beta-lactam antibiotics. Clin Infect Dis. 1997 Jan;24 (Suppl 1):S19–S45. doi: 10.1093/clinids/24.supplement_1.s19. [DOI] [PubMed] [Google Scholar]
  18. Petrosino J. F., Palzkill T. Systematic mutagenesis of the active site omega loop of TEM-1 beta-lactamase. J Bacteriol. 1996 Apr;178(7):1821–1828. doi: 10.1128/jb.178.7.1821-1828.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Prinarakis E. E., Miriagou V., Tzelepi E., Gazouli M., Tzouvelekis L. S. Emergence of an inhibitor-resistant beta-lactamase (SHV-10) derived from an SHV-5 variant. Antimicrob Agents Chemother. 1997 Apr;41(4):838–840. doi: 10.1128/aac.41.4.838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Saves I., Burlet-Schiltz O., Maveyraud L., Samama J. P., Promé J. C., Masson J. M. Mass spectral kinetic study of acylation and deacylation during the hydrolysis of penicillins and cefotaxime by beta-lactamase TEM-1 and the G238S mutant. Biochemistry. 1995 Sep 19;34(37):11660–11667. doi: 10.1021/bi00037a003. [DOI] [PubMed] [Google Scholar]
  21. Vecoli C., Prevost F. E., Ververis J. J., Medeiros A. A., O'Leary G. P., Jr Comparison of polyacrylamide and agarose gel thin-layer isoelectric focusing for the characterization of beta-lactamases. Antimicrob Agents Chemother. 1983 Aug;24(2):186–189. doi: 10.1128/aac.24.2.186. [DOI] [PMC free article] [PubMed] [Google Scholar]

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