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. 1991 Sep 15;278(Pt 3):673–678. doi: 10.1042/bj2780673

The mutation Lys234His yields a class A beta-lactamase with a novel pH-dependence.

J Brannigan 1, A Matagne 1, F Jacob 1, C Damblon 1, B Joris 1, D Klein 1, B G Spratt 1, J M Frère 1
PMCID: PMC1151399  PMID: 1910335

Abstract

The lysine-234 residue is highly conserved in beta-lactamases and in nearly all active-site-serine penicillin-recognizing enzymes. Its replacement by a histidine residue in the Streptomyces albus G class A beta-lactamase yielded an enzyme the pH-dependence of which was characterized by the appearance of a novel pK, which could be attributed to the newly introduced residue. At low pH, the kcat, value for benzylpenicillin was as high as 50% of that of the wild-type enzyme, demonstrating that an efficient active site was maintained. Both kcat. and kcat/Km dramatically decreased above pH 6 but the decrease in kcat./Km could not be attributed to larger Km values. Thus a positive charge on the side chain of residue 234 appears to be more essential for transition-state stabilization than for initial recognition of the substrate ground state.

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Selected References

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

  1. Adam M., Damblon C., Plaitin B., Christiaens L., Frère J. M. Chromogenic depsipeptide substrates for beta-lactamases and penicillin-sensitive DD-peptidases. Biochem J. 1990 Sep 1;270(2):525–529. doi: 10.1042/bj2700525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Dehottay P., Dusart J., Duez C., Lenzini M. V., Martial J. A., Frère J. M., Ghuysen J. M., Kieser T. Cloning and amplified expression in Streptomyces lividans of a gene encoding extracellular beta-lactamase from Streptomyces albus G. Gene. 1986;42(1):31–36. doi: 10.1016/0378-1119(86)90147-2. [DOI] [PubMed] [Google Scholar]
  5. Ellerby L. M., Escobar W. A., Fink A. L., Mitchinson C., Wells J. A. The role of lysine-234 in beta-lactamase catalysis probed by site-directed mutagenesis. Biochemistry. 1990 Jun 19;29(24):5797–5806. doi: 10.1021/bi00476a022. [DOI] [PubMed] [Google Scholar]
  6. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Jacob F., Joris B., Dideberg O., Dusart J., Ghuysen J. M., Frère J. M. Engineering a novel beta-lactamase by a single point mutation. Protein Eng. 1990 Oct;4(1):79–86. doi: 10.1093/protein/4.1.79. [DOI] [PubMed] [Google Scholar]
  9. Jacob F., Joris B., Lepage S., Dusart J., Frère J. M. Role of the conserved amino acids of the 'SDN' loop (Ser130, Asp131 and Asn132) in a class A beta-lactamase studied by site-directed mutagenesis. Biochem J. 1990 Oct 15;271(2):399–406. doi: 10.1042/bj2710399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Kelly J. A., Knox J. R., Zhao H., Frère J. M., Ghaysen J. M. Crystallographic mapping of beta-lactams bound to a D-alanyl-D-alanine peptidase target enzyme. J Mol Biol. 1989 Sep 20;209(2):281–295. doi: 10.1016/0022-2836(89)90277-5. [DOI] [PubMed] [Google Scholar]
  12. Kramer W., Drutsa V., Jansen H. W., Kramer B., Pflugfelder M., Fritz H. J. The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 1984 Dec 21;12(24):9441–9456. doi: 10.1093/nar/12.24.9441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Matagne A., Joris B., Van Beeumen J., Frère J. M. Ragged N-termini and other variants of class A beta-lactamases analysed by chromatofocusing. Biochem J. 1991 Feb 1;273(Pt 3):503–510. doi: 10.1042/bj2730503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Matagne A., Misselyn-Bauduin A. M., Joris B., Erpicum T., Granier B., Frère J. M. The diversity of the catalytic properties of class A beta-lactamases. Biochem J. 1990 Jan 1;265(1):131–146. doi: 10.1042/bj2650131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Waley S. G. The pH-dependence and group modification of beta-lactamase I. Biochem J. 1975 Sep;149(3):547–551. doi: 10.1042/bj1490547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]

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