Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1994 Aug 15;302(Pt 1):1–4. doi: 10.1042/bj3020001

The role of lysine-67 in a class C beta-lactamase is mainly electrostatic.

D Monnaie 1, A Dubus 1, J M Frère 1
PMCID: PMC1137182  PMID: 8067994

Abstract

By using site-directed mutagenesis, the conserved Lys-67 residue situated three positions after the active-site Ser of a class C beta-lactamase was replaced by Arg or Gln. The Lys-67-Gln protein was nearly inactive. Although severely impaired, the Lys-67-Arg mutant exhibited an appreciable activity above pH 7.5 and, for some poor substrates of the wild-type enzyme, the kcat. values were even increased. The properties of the Lys-67-Arg mutant were studied by both steady-state and transient-state kinetic methods with a variety of compounds representing distinct classes of available substrates. With beta-lactam substrates, the kcat./Km values reflecting the efficiency of the acylation step (k+2/K) were decreased 25-100-fold. When the individual values could be measured, k+2 was not significantly altered, but K was found to be strongly increased, a result most likely explained by a corresponding increase in the k+1/k-1 ratio. These results, combined with the much stronger impairment of the Lys-67-Gln mutant, can be interpreted by attributing an electrostatic role to the positive ammonium group of the Lys-67 side chain.

Full text

PDF
1

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brannigan J., Matagne A., Jacob F., Damblon C., Joris B., Klein D., Spratt B. G., Frère J. M. The mutation Lys234His yields a class A beta-lactamase with a novel pH-dependence. Biochem J. 1991 Sep 15;278(Pt 3):673–678. doi: 10.1042/bj2780673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. De Meester F., Joris B., Reckinger G., Bellefroid-Bourguignon C., Frère J. M., Waley S. G. Automated analysis of enzyme inactivation phenomena. Application to beta-lactamases and DD-peptidases. Biochem Pharmacol. 1987 Jul 15;36(14):2393–2403. doi: 10.1016/0006-2952(87)90609-5. [DOI] [PubMed] [Google Scholar]
  3. Dubus A., Monnaie D., Jacobs C., Normark S., Frère J. M. A dramatic change in the rate-limiting step of beta-lactam hydrolysis results from the substitution of the active-site serine residue by a cysteine in the class-C beta-lactamase of Enterobacter cloacae 908R. Biochem J. 1993 Jun 1;292(Pt 2):537–543. doi: 10.1042/bj2920537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Galleni M., Amicosante G., Frère J. M. A survey of the kinetic parameters of class C beta-lactamases. Cephalosporins and other beta-lactam compounds. Biochem J. 1988 Oct 1;255(1):123–129. doi: 10.1042/bj2550123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Galleni M., Frère J. M. A survey of the kinetic parameters of class C beta-lactamases. Penicillins. Biochem J. 1988 Oct 1;255(1):119–122. doi: 10.1042/bj2550119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gibson R. M., Christensen H., Waley S. G. Site-directed mutagenesis of beta-lactamase I. Single and double mutants of Glu-166 and Lys-73. Biochem J. 1990 Dec 15;272(3):613–619. doi: 10.1042/bj2720613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hadonou A. M., Wilkin J. M., Varetto L., Joris B., Lamotte-Brasseur J., Klein D., Duez C., Ghuysen J. M., Frère J. M. Site-directed mutagenesis of the Streptomyces R61 DD-peptidase. Catalytic function of the conserved residues around the active site and a comparison with class-A and class-C beta-lactamases. Eur J Biochem. 1992 Jul 1;207(1):97–102. doi: 10.1111/j.1432-1033.1992.tb17025.x. [DOI] [PubMed] [Google Scholar]
  8. Herzberg O., Moult J. Bacterial resistance to beta-lactam antibiotics: crystal structure of beta-lactamase from Staphylococcus aureus PC1 at 2.5 A resolution. Science. 1987 May 8;236(4802):694–701. doi: 10.1126/science.3107125. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Knott-Hunziker V., Petursson S., Waley S. G., Jaurin B., Grundström T. The acyl-enzyme mechanism of beta-lactamase action. The evidence for class C Beta-lactamases. Biochem J. 1982 Nov 1;207(2):315–322. doi: 10.1042/bj2070315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lamotte-Brasseur J., Dive G., Dideberg O., Charlier P., Frère J. M., Ghuysen J. M. Mechanism of acyl transfer by the class A serine beta-lactamase of Streptomyces albus G. Biochem J. 1991 Oct 1;279(Pt 1):213–221. doi: 10.1042/bj2790213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lobkovsky E., Moews P. C., Liu H., Zhao H., Frere J. M., Knox J. R. Evolution of an enzyme activity: crystallographic structure at 2-A resolution of cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11257–11261. doi: 10.1073/pnas.90.23.11257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Matagne A., Lamotte-Brasseur J., Dive G., Knox J. R., Frère J. M. Interactions between active-site-serine beta-lactamases and compounds bearing a methoxy side chain on the alpha-face of the beta-lactam ring: kinetic and molecular modelling studies. Biochem J. 1993 Aug 1;293(Pt 3):607–611. doi: 10.1042/bj2930607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Monnaie D., Dubus A., Cooke D., Marchand-Brynaert J., Normark S., Frère J. M. Role of residue Lys315 in the mechanism of action of the Enterobacter cloacae 908R beta-lactamase. Biochemistry. 1994 May 3;33(17):5193–5201. doi: 10.1021/bi00183a024. [DOI] [PubMed] [Google Scholar]
  15. Monnaie D., Virden R., Frère J. M. A rapid-kinetic study of the class C beta-lactamase of Enterobacter cloacae 908R. FEBS Lett. 1992 Jul 20;306(2-3):108–112. doi: 10.1016/0014-5793(92)80979-q. [DOI] [PubMed] [Google Scholar]
  16. Oefner C., D'Arcy A., Daly J. J., Gubernator K., Charnas R. L., Heinze I., Hubschwerlen C., Winkler F. K. Refined crystal structure of beta-lactamase from Citrobacter freundii indicates a mechanism for beta-lactam hydrolysis. Nature. 1990 Jan 18;343(6255):284–288. doi: 10.1038/343284a0. [DOI] [PubMed] [Google Scholar]
  17. Strynadka N. C., Adachi H., Jensen S. E., Johns K., Sielecki A., Betzel C., Sutoh K., James M. N. Molecular structure of the acyl-enzyme intermediate in beta-lactam hydrolysis at 1.7 A resolution. Nature. 1992 Oct 22;359(6397):700–705. doi: 10.1038/359700a0. [DOI] [PubMed] [Google Scholar]
  18. Tsukamoto K., Tachibana K., Yamazaki N., Ishii Y., Ujiie K., Nishida N., Sawai T. Role of lysine-67 in the active site of class C beta-lactamase from Citrobacter freundii GN346. Eur J Biochem. 1990 Feb 22;188(1):15–22. doi: 10.1111/j.1432-1033.1990.tb15365.x. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES