Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 May 1;331(Pt 3):703–711. doi: 10.1042/bj3310703

The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent beta-lactamase.

S Bounaga 1, A P Laws 1, M Galleni 1, M I Page 1
PMCID: PMC1219408  PMID: 9560295

Abstract

The plot of kcat/Km against pH for the Bacillus cereus 569/H beta-lactamase class B catalysed hydrolysis of benzylpenicillin and cephalosporin indicates that there are three catalytically important groups, two of pKa 5.6+/-0.2 and one of pKa 9.5+/-0.2. Below pH 5 there is an inverse second-order dependence of reactivity upon hydrogen ion concentration, indicative of the requirement of two basic residues for catalysis. These are assigned to zinc(II)-bound water and Asp-90, both with a pKa of 5.6+/-0.2. A thiol, N-(2'-mercaptoethyl)-2-phenylacetamide, is an inhibitor of the class B enzyme with a Ki of 70 microM. The pH-dependence of Ki shows similar pH inflections to those observed in the catalysed hydrolysis of substrates. The pH-independence of Ki between pH 6 and 9 indicates that the pKa of zinc(II)-bound water must be 5.6 and not the higher pKa of 9.5. The kinetic solvent isotope effect on kcat/Km is 1.3+/-0.5 and that on kcat is 1.5. There is no effect on reactivity by either added zinc(II) or methanol. The possible mechanisms of action for the class B beta-lactamase are discussed, and it is concluded that zinc(II) acts as a Lewis acid to stabilize the dianionic form of the tetrahedral intermediate and to provide a hydroxide-ion bound nucleophile, whereas the carboxylate anion of Asp-90 acts as a general base to form the dianion and also, presumably, as a general acid catalyst facilitating C-N bond fission.

Full Text

The Full Text of this article is available as a PDF (469.9 KB).

Selected References

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

  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., Daniel M., Fleming J., Hermoso J. M., Pang C., Waley S. G. The amino acid sequence of the zinc-requiring beta-lactamase II from the bacterium Bacillus cereus 569. FEBS Lett. 1985 Sep 23;189(2):207–211. doi: 10.1016/0014-5793(85)81024-3. [DOI] [PubMed] [Google Scholar]
  3. Baldwin G. S., Galdes A., Hill H. A., Smith B. E., Waley S. G., Abraham E. P. Histidine residues of zinc ligands in beta-lactamase II. Biochem J. 1978 Nov 1;175(2):441–447. doi: 10.1042/bj1750441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Banci L., Bertini I., La Penna G. The enzymatic mechanism of carboxypeptidase: a molecular dynamics study. Proteins. 1994 Feb;18(2):186–197. doi: 10.1002/prot.340180210. [DOI] [PubMed] [Google Scholar]
  5. Bandoh K., Muto Y., Watanabe K., Katoh N., Ueno K. Biochemical properties and purification of metallo-beta-lactamase from Bacteroides fragilis. Antimicrob Agents Chemother. 1991 Feb;35(2):371–372. doi: 10.1128/aac.35.2.371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bicknell R., Emanuel E. L., Gagnon J., Waley S. G. The production and molecular properties of the zinc beta-lactamase of Pseudomonas maltophilia IID 1275. Biochem J. 1985 Aug 1;229(3):791–797. doi: 10.1042/bj2290791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bicknell R., Knott-Hunziker V., Waley S. G. The pH-dependence of class B and class C beta-lactamases. Biochem J. 1983 Jul 1;213(1):61–66. doi: 10.1042/bj2130061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Breslow R., Chin J., Hilvert D., Trainor G. Evidence for the general base mechanism in carboxypeptidase A-catalyzed reactions: partitioning studies on nucleophiles and H2(18)O kinetic isotope effects. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4585–4589. doi: 10.1073/pnas.80.14.4585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Carfi A., Pares S., Duée E., Galleni M., Duez C., Frère J. M., Dideberg O. The 3-D structure of a zinc metallo-beta-lactamase from Bacillus cereus reveals a new type of protein fold. EMBO J. 1995 Oct 16;14(20):4914–4921. doi: 10.1002/j.1460-2075.1995.tb00174.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Carfí A., Paul-Soto R., Martin L., Pétillot Y., Frère J. M., Dideberg O. Purification, crystallization and preliminary X-ray analysis of Bacteroides fragilis Zn2+ beta-lactamase. Acta Crystallogr D Biol Crystallogr. 1997 Jul 1;53(Pt 4):485–487. doi: 10.1107/S0907444997000966. [DOI] [PubMed] [Google Scholar]
  11. Christianson D. W., David P. R., Lipscomb W. N. Mechanism of carboxypeptidase A: hydration of a ketonic substrate analogue. Proc Natl Acad Sci U S A. 1987 Mar;84(6):1512–1515. doi: 10.1073/pnas.84.6.1512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cleland W. W. Determining the chemical mechanisms of enzyme-catalyzed reactions by kinetic studies. Adv Enzymol Relat Areas Mol Biol. 1977;45:273–387. doi: 10.1002/9780470122907.ch4. [DOI] [PubMed] [Google Scholar]
  13. Concha N. O., Rasmussen B. A., Bush K., Herzberg O. Crystal structure of the wide-spectrum binuclear zinc beta-lactamase from Bacteroides fragilis. Structure. 1996 Jul 15;4(7):823–836. doi: 10.1016/s0969-2126(96)00089-5. [DOI] [PubMed] [Google Scholar]
  14. Crowder M. W., Wang Z., Franklin S. L., Zovinka E. P., Benkovic S. J. Characterization of the metal-binding sites of the beta-lactamase from Bacteroides fragilis. Biochemistry. 1996 Sep 17;35(37):12126–12132. doi: 10.1021/bi960976h. [DOI] [PubMed] [Google Scholar]
  15. Cuchural G. J., Jr, Malamy M. H., Tally F. P. Beta-lactamase-mediated imipenem resistance in Bacteroides fragilis. Antimicrob Agents Chemother. 1986 Nov;30(5):645–648. doi: 10.1128/aac.30.5.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dale J. W., Godwin D., Mossakowska D., Stephenson P., Wall S. Sequence of the OXA2 beta-lactamase: comparison with other penicillin-reactive enzymes. FEBS Lett. 1985 Oct 21;191(1):39–44. doi: 10.1016/0014-5793(85)80989-3. [DOI] [PubMed] [Google Scholar]
  17. Davies R. B., Abraham E. P. Metal cofactor requirements of beta-lactamase II. Biochem J. 1974 Oct;143(1):129–135. doi: 10.1042/bj1430129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Felici A., Amicosante G., Oratore A., Strom R., Ledent P., Joris B., Fanuel L., Frère J. M. An overview of the kinetic parameters of class B beta-lactamases. Biochem J. 1993 Apr 1;291(Pt 1):151–155. doi: 10.1042/bj2910151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hussain M., Carlino A., Madonna M. J., Lampen J. O. Cloning and sequencing of the metallothioprotein beta-lactamase II gene of Bacillus cereus 569/H in Escherichia coli. J Bacteriol. 1985 Oct;164(1):223–229. doi: 10.1128/jb.164.1.223-229.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Iaconis J. P., Sanders C. C. Purification and characterization of inducible beta-lactamases in Aeromonas spp. Antimicrob Agents Chemother. 1990 Jan;34(1):44–51. doi: 10.1128/aac.34.1.44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jaurin B., Grundström T. ampC cephalosporinase of Escherichia coli K-12 has a different evolutionary origin from that of beta-lactamases of the penicillinase type. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4897–4901. doi: 10.1073/pnas.78.8.4897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Joris B., Ledent P., Dideberg O., Fonzé E., Lamotte-Brasseur J., Kelly J. A., Ghuysen J. M., Frère J. M. Comparison of the sequences of class A beta-lactamases and of the secondary structure elements of penicillin-recognizing proteins. Antimicrob Agents Chemother. 1991 Nov;35(11):2294–2301. doi: 10.1128/aac.35.11.2294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kiefer L. L., Fierke C. A. Functional characterization of human carbonic anhydrase II variants with altered zinc binding sites. Biochemistry. 1994 Dec 27;33(51):15233–15240. doi: 10.1021/bi00255a003. [DOI] [PubMed] [Google Scholar]
  24. Lim H. M., Iyer R. K., Pène J. J. Site-directed mutagenesis of dicarboxylic acids near the active site of Bacillus cereus 5/B/6 beta-lactamase II. Biochem J. 1991 Jun 1;276(Pt 2):401–404. doi: 10.1042/bj2760401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lim H. M., Pène J. J. Mutations affecting the catalytic activity of Bacillus cereus 5/B/6 beta-lactamase II. J Biol Chem. 1989 Jul 15;264(20):11682–11687. [PubMed] [Google Scholar]
  26. Lim H. M., Pène J. J., Shaw R. W. Cloning, nucleotide sequence, and expression of the Bacillus cereus 5/B/6 beta-lactamase II structural gene. J Bacteriol. 1988 Jun;170(6):2873–2878. doi: 10.1128/jb.170.6.2873-2878.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Little C., Emanuel E. L., Gagnon J., Waley S. G. Identification of an essential glutamic acid residue in beta-lactamase II from Bacillus cereus. Biochem J. 1986 Jan 15;233(2):465–469. doi: 10.1042/bj2330465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Massidda O., Rossolini G. M., Satta G. The Aeromonas hydrophila cphA gene: molecular heterogeneity among class B metallo-beta-lactamases. J Bacteriol. 1991 Aug;173(15):4611–4617. doi: 10.1128/jb.173.15.4611-4617.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Medeiros A. A. Beta-lactamases. Br Med Bull. 1984 Jan;40(1):18–27. doi: 10.1093/oxfordjournals.bmb.a071942. [DOI] [PubMed] [Google Scholar]
  30. Mock W. L., Tsay J. T. A probe of the active site acidity of carboxypeptidase A. Biochemistry. 1986 May 20;25(10):2920–2927. doi: 10.1021/bi00358a028. [DOI] [PubMed] [Google Scholar]
  31. Mock W. L., Tsay J. T. pK values for active site residues of carboxypeptidase A. J Biol Chem. 1988 Jun 25;263(18):8635–8641. [PubMed] [Google Scholar]
  32. Mustafi D., Makinen M. W. Catalytic conformation of carboxypeptidase A. Structure of a true enzyme reaction intermediate determined by electron nuclear double resonance. J Biol Chem. 1994 Feb 11;269(6):4587–4595. [PubMed] [Google Scholar]
  33. Osumi A., Rahmo A., King S. W., Przystas T. J., Fife T. H. Substituent effects in the carboxypeptidase A catalyzed hydrolysis of substituted L,beta-phenyllactate esters. Biochemistry. 1994 Dec 13;33(49):14750–14757. doi: 10.1021/bi00253a013. [DOI] [PubMed] [Google Scholar]
  34. Payne D. J. Metallo-beta-lactamases--a new therapeutic challenge. J Med Microbiol. 1993 Aug;39(2):93–99. doi: 10.1099/00222615-39-2-93. [DOI] [PubMed] [Google Scholar]
  35. Pocker Y., Bjorkquist D. W. Comparative studies of bovine carbonic anhydrase in H2O and D2O. Stopped-flow studies of the kinetics of interconversion of CO2 and HCO3. Biochemistry. 1977 Dec 27;16(26):5698–5707. doi: 10.1021/bi00645a008. [DOI] [PubMed] [Google Scholar]
  36. Rasmussen B. A., Gluzman Y., Tally F. P. Cloning and sequencing of the class B beta-lactamase gene (ccrA) from Bacteroides fragilis TAL3636. Antimicrob Agents Chemother. 1990 Aug;34(8):1590–1592. doi: 10.1128/aac.34.8.1590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rees D. C., Lewis M., Lipscomb W. N. Refined crystal structure of carboxypeptidase A at 1.54 A resolution. J Mol Biol. 1983 Aug 5;168(2):367–387. doi: 10.1016/s0022-2836(83)80024-2. [DOI] [PubMed] [Google Scholar]
  38. Suh J., Kaiser E. T. pH dependence of the nitrotyrosine-248 and arsanilazotyrosine-248 carboxypeptidase A catalyzed hydrolysis of O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate. J Am Chem Soc. 1976 Mar 31;98(7):1940–1947. doi: 10.1021/ja00423a048. [DOI] [PubMed] [Google Scholar]
  39. Sutton B. J., Artymiuk P. J., Cordero-Borboa A. E., Little C., Phillips D. C., Waley S. G. An X-ray-crystallographic study of beta-lactamase II from Bacillus cereus at 0.35 nm resolution. Biochem J. 1987 Nov 15;248(1):181–188. doi: 10.1042/bj2480181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sykes R. B., Matthew M. The beta-lactamases of gram-negative bacteria and their role in resistance to beta-lactam antibiotics. J Antimicrob Chemother. 1976 Jun;2(2):115–157. doi: 10.1093/jac/2.2.115. [DOI] [PubMed] [Google Scholar]
  41. Thompson J. S., Malamy M. H. Sequencing the gene for an imipenem-cefoxitin-hydrolyzing enzyme (CfiA) from Bacteroides fragilis TAL2480 reveals strong similarity between CfiA and Bacillus cereus beta-lactamase II. J Bacteriol. 1990 May;172(5):2584–2593. doi: 10.1128/jb.172.5.2584-2593.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Xiang S., Short S. A., Wolfenden R., Carter C. W., Jr Cytidine deaminase complexed to 3-deazacytidine: a "valence buffer" in zinc enzyme catalysis. Biochemistry. 1996 Feb 6;35(5):1335–1341. doi: 10.1021/bi9525583. [DOI] [PubMed] [Google Scholar]

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

RESOURCES