Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1987 Oct 1;247(1):29–33. doi: 10.1042/bj2470029

Inactivation of the thiol RTEM-1 beta-lactamase by 6-beta-bromopenicillanic acid. Identity of the primary active-site nucleophile.

A K Knap 1, R F Pratt 1
PMCID: PMC1148364  PMID: 2825657

Abstract

The thiol RTEM-1 beta-lactamase [Sigal, Harwood & Arentzen (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7157-7160] is inactivated by 6-beta-bromopenicillanic acid with formation of a characteristic chromophore, absorbing maximally at 350 nm, which is covalently bound to the enzyme. Model studies suggest that the chromophore is that of a 6-carboxylate thiol ester of 2,3-dihydro-2,2-dimethyl-1,4-thiazine-3,6-dicarboxylate, which can arise by rearrangement of the thiol-penicilloate obtained by thiolysis of the beta-lactam of 6-beta-bromopenicillanate. Loss of activity of the enzyme is also concerted with disappearance of its single (cysteine) thiol group. These results indicate that the thiol group of this enzyme is indeed a nucleophilic catalyst in beta-lactam turnover. The thiol beta-lactamase is also inactivated by clavulanic acid with formation of a chromophore, presumably a 3-aminoacrylate thiol ester, at 308 nm. Both 6-beta-bromopenicillanate and clavulanate are hydrolysed more slowly by the thiol enzyme than by the native serine beta-lactamase, but, probably as a consequence of this, both compounds inactivate the former enzyme more efficiently. Cefoxitin, a poor substrate of the native enzyme, does not appear to interact covalently with the thiol beta-lactamase.

Full text

PDF
33

Selected References

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

  1. 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]
  2. Brenner D. G., Knowles J. R. Penicillanic acid sulfone: nature of irreversible inactivation of RTEM beta-lactamase from Escherichia coli. Biochemistry. 1984 Nov 20;23(24):5833–5839. doi: 10.1021/bi00319a024. [DOI] [PubMed] [Google Scholar]
  3. Brocklehurst K., Little G. Reactions of papain and of low-molecular-weight thiols with some aromatic disulphides. 2,2'-Dipyridyl disulphide as a convenient active-site titrant for papain even in the presence of other thiols. Biochem J. 1973 May;133(1):67–80. doi: 10.1042/bj1330067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brocklehurst K., Malthouse J. P. Evidence that the lack of high catalytic activity of thiolsubtilisin towards specific substrates may be due to an inappropriately located proton-distribution system. Demonstration of highly nucleophilic character of the thiol group of thiolsubtilisin in the catalytically relevant ionization state of the active centre by use of a two-protonic-state reactivity probe. Biochem J. 1981 Mar 1;193(3):819–823. doi: 10.1042/bj1930819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Charnas R. L., Knowles J. R. Inactivation of RTEM beta-lactamase from Escherichia coli by clavulanic acid and 9-deoxyclavulanic acid. Biochemistry. 1981 May 26;20(11):3214–3219. doi: 10.1021/bi00514a035. [DOI] [PubMed] [Google Scholar]
  6. Cohen S. A., Pratt R. F. Inactivation of Bacillus cereus beta-lactamase I by 6 beta-bromopencillanic acid: mechanism. Biochemistry. 1980 Aug 19;19(17):3996–4003. doi: 10.1021/bi00558a017. [DOI] [PubMed] [Google Scholar]
  7. De Meester F., Frère J. M., Waley S. G., Cartwright S. J., Virden R., Lindberg F. 6-beta-Iodopenicillanate as a probe for the classification of beta-lactamases. Biochem J. 1986 Nov 1;239(3):575–580. doi: 10.1042/bj2390575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fisher J., Belasco J. G., Khosla S., Knowles J. R. beta-Lactamase proceeds via an acyl-enzyme intermediate. Interaction of the Escherichia coli RTEM enzyme with cefoxitin. Biochemistry. 1980 Jun 24;19(13):2895–2901. doi: 10.1021/bi00554a012. [DOI] [PubMed] [Google Scholar]
  9. Fisher J., Charnas R. L., Knowles J. R. Kinetic studies on the inactivation of Escherichia coli RTEM beta-lactamase by clavulanic acid. Biochemistry. 1978 May 30;17(11):2180–2184. doi: 10.1021/bi00604a024. [DOI] [PubMed] [Google Scholar]
  10. Hardy L. W., Kirsch J. F. Diffusion-limited component of reactions catalyzed by Bacillus cereus beta-lactamase I. Biochemistry. 1984 Mar;23(6):1275–1282. doi: 10.1021/bi00301a040. [DOI] [PubMed] [Google Scholar]
  11. Joris B., Dusart J., Frere J. M., van Beeumen J., Emanuel E. L., Petursson S., Gagnon J., Waley S. G. The active site of the P99 beta-lactamase from Enterobacter cloacae. Biochem J. 1984 Oct 1;223(1):271–274. doi: 10.1042/bj2230271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Knott-Hunziker V., Waley S. G., Orlek B. S., Sammes P. G. Penicillinase active sites: labelling of serine-44 in beta-lactamase I by 6beta-bromopenicillanic acid. FEBS Lett. 1979 Mar 1;99(1):59–61. doi: 10.1016/0014-5793(79)80248-3. [DOI] [PubMed] [Google Scholar]
  14. Loosemore M. J., Cohen S. A., Pratt R. F. Inactivation of Bacillus cereus beta-lactamase I by 6 beta-bromopenicillanic acid: kinetics. Biochemistry. 1980 Aug 19;19(17):3990–3995. doi: 10.1021/bi00558a016. [DOI] [PubMed] [Google Scholar]
  15. Neet K. E., Nanci A., Koshland D. E., Jr Properties of thiol-subtilisin. The consequences of converting the active serine residue to cysteine in a serine protease. J Biol Chem. 1968 Dec 25;243(24):6392–6401. [PubMed] [Google Scholar]
  16. Philipp M., Tsai I. H., Bender M. L. Comparison of the kinetic specificity of subtilisin and thiolsubtilisin toward n-alkyl p-nitrophenyl esters. Biochemistry. 1979 Aug 21;18(17):3769–3773. doi: 10.1021/bi00584a020. [DOI] [PubMed] [Google Scholar]
  17. Polgár L., Bender M. L. Simulated mutation at the active site of biologically active proteins. Adv Enzymol Relat Areas Mol Biol. 1970;33:381–400. doi: 10.1002/9780470122785.ch8. [DOI] [PubMed] [Google Scholar]
  18. Pratt R. F., Loosemore M. J. 6-beta-bromopenicillanic acid, a potent beta-lactamase inhibitor. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4145–4149. doi: 10.1073/pnas.75.9.4145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sigal I. S., DeGrado W. F., Thomas B. J., Petteway S. R., Jr Purification and properties of thiol beta-lactamase. A mutant of pBR322 beta-lactamase in which the active site serine has been replaced with cysteine. J Biol Chem. 1984 Apr 25;259(8):5327–5332. [PubMed] [Google Scholar]
  20. Sigal I. S., Harwood B. G., Arentzen R. Thiol-beta-lactamase: replacement of the active-site serine of RTEM beta-lactamase by a cysteine residue. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7157–7160. doi: 10.1073/pnas.79.23.7157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tsai I. H., Bender M. L. Conformation of the active site of thiolsubtilisin: reaction with specific chloromethyl ketones and arylacryloylimidazoles. Biochemistry. 1979 Aug 21;18(17):3764–3768. doi: 10.1021/bi00584a019. [DOI] [PubMed] [Google Scholar]
  22. Yokosawa H., Ojima S., Ishii S. Thioltrypsin. Chemical transformation of the active-site serine residue of Streptomyces griseus trypsin to a cysteine residue. J Biochem. 1977 Sep;82(3):869–876. doi: 10.1093/oxfordjournals.jbchem.a131763. [DOI] [PubMed] [Google Scholar]

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

RESOURCES