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Enzyme deactivation due to metal-ion dissociation during turnover of the cobalt β-lactamase catalysed hydrolysis of β-lactams

Badarau, Adriana and Page, Michael I. (2006) Enzyme deactivation due to metal-ion dissociation during turnover of the cobalt β-lactamase catalysed hydrolysis of β-lactams. Biochemistry, 45 (35). pp. 11012-11020. ISSN 0006-2960

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Metallo-β-lactamases are native zinc enzymes that catalyze the hydrolysis of -lactam antibiotics but are also able to function with cobalt (II) and require one or two metal ions for catalytic activity. The kinetics of the hydrolysis of benzylpenicillin catalyzed by cobalt substituted -lactamase from Bacillus cereus (BcII) are biphasic. The dependence of enzyme activity on pH and metal-ion concentration indicates that only the di-cobalt enzyme is catalytically active. A mono-cobalt enzyme species is formed during the catalytic cycle, which is virtually inactive and requires the association of another cobalt ion for turnover. Two intermediates with different metal to enzyme stoichiometries are formed on a branched reaction pathway. The di-cobalt enzyme intermediate is responsible for the direct catalytic route, which is pH-independent between 5.5 and 9.5 but is also able to slowly lose one bound cobalt ion via the branching route to give the mono-cobalt inactive enzyme intermediate. This inactivation pathway of metal-ion dissociation occurs by both an acid catalyzed and a pH-independent reaction, which is dependent on the presence of an enzyme residue of pKa = 8.9 ± 0.1 in its protonated form and shows a large kinetic solvent isotope effect (H2O/D2O) of 5.2 ± 0.5, indicative of a rate-limiting proton transfer. The pseudo first-order rate constant to regenerate the di-cobalt -lactamase from the mono-cobalt enzyme intermediate has a first-order dependence on cobalt-ion concentration in the pH range 5.5-9.5. The second-order rate constant for metal-ion association is dependent on two groups of pKa 6.32 ± 0.1 and 7.47 ± 0.1 being in their deprotonated basic forms and one group of pKa 9.48 ± 0.1 being in its protonated form

Item Type: Article
Additional Information: UoA 18 (Chemistry)
Subjects: Q Science > QD Chemistry
Schools: School of Applied Sciences
School of Applied Sciences > Biomolecular Sciences Research Centre
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References: 1. Fre`re, J. M. (1995) Beta-lactamases and bacterial resistance to antibiotics, Mol. Microbiol. 16, 385-395. 2. Galleni, M., Lamotte-Brasseur, J., Rossolini, G. M., Spencer, J., Dideberg, O., and Fre`re, J. M. (2001) Standard numbering scheme for class B â-lactamases, Antimicrob. Agents Chemother. 45, 660- 663. 3. Fabiane, S. M., Sohi, M. K., Wan, T., Payne, D. J., Bateson, J. H., Mitchell, T., and Sutton, B. J. (1998) Crystal structure of the zinc-dependent â-lactamase from Bacillus cereus at 1.9 Å resolution: binuclear active site with features of a mononuclear enzyme, Biochemistry 37, 12404-12411 4. Orellano, E. G., Girardini, J. E., Cricco, J. A., Ceccarelli, E. A., and Vila, A. J. (1998) Spectroscopic characterization of a binuclear metal site in Bacillus cereus â-lactamase II, Biochemistry 37, 10173-10180. 5. Paul-Soto, R., Bauer, R., Fre`re, J. M., Galleni, M., Meyer-Klaucke, W., Nolting, H., Rossolini, G. M., de Seny, D., Hernandez- Valladares, M., Zeppezauer, M., and Adolph, H. W. (1999) Monoand binuclear Zn2+-â-lactamase. Role of the conserved cysteine in the catalytic mechanism, J. Biol. Chem. 274, 13242-13249. 6. Concha, N. O., Rasmussen, B. A., Bush, K., and Herzberg, O. (1996) Crystal structure of the wide-spectrum binuclear zinc â-lactamase from Bacteroides fragilis, Structure 4, 823-836. 7. Paul-Soto, R., Hernadez-Valladares, M., Galleni, M., Bauer, R., Zeppezauer, M., Fre`re, J. M., and Adolph, H. W. (1998) Monoand binuclear Zn2+-â-lactamase from Bacteroides fragilis: catalytic and structural roles of the zinc ions, FEBS Lett. 438, 137- 140. 8. Yang, Y., Keeney, D., Tang, X., Canfield, N., and Rasmussen, B. A. (1999) Kinetic properties and metal content of the metallo-â -lactamase CcrA harboring selective amino acid substitutions, J. Biol. Chem. 274, 15706-15711. 9. Wang, Z., Fast, W., and Benkovic, S. J. (1999) On the mechanism of the Bacteroides fragilis metallo-â-lactamase, Biochemistry 38, 10013-10023. 10. Laraki, N., Franceschini, N., Rossolini, G. M., Santucci, P., Meunier, C., de Pauw, E., Amicosante, G., Fre`re, J. M., and Galleni, M. (1999) Biochemical characterisation of the Pseudomonas aeruginosa 101/1477 metallo-â-lactamase IMP-1 produced by Escherichia coli, Antimicrob. Agents Chemother. 43, 902- 906. 11. Haruta, S., Yamaguchi, H., Yamamoto, E. T., Eriguchi, Y., Nukaga, M., O’Hara, K., and Sawai, T. (2000) Functional analysis of the active site of a metallo-â-lactamase proliferating in Japan, Antimicrob. Agents Chemother. 44, 2304-2309. 12. Concha, N. O., Janson, C. A., Rowling, P., Pearson, S., Cheever, C. A., Clarke, B. P., Lewis, C., Galleni, M., Fre`re, J. M., Payne, D. J., Bateson, J. H., and Abdel-Meguid, S. S. (2000) Crystal structure of the IMP-1 metallo â-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor: binding determinants of a potent, broad-spectrum inhibitor, Biochemistry 39, 4288-4298. 13. Garcia-Saez, I., Hopkins, J., Papamicael, C., Franceschini, N., Amicosante, G., Rossolini, G. M., Galleni, M., Fre`re, J. M., and Dideberg, O. (2003) The 1.5-A Structure of Chryseobacterium meningosepticum zinc â-Lactamase in complex with the inhibitor, D-captopril, J. Biol. Chem. 278, 23868-23873. 14. Crowder, M. W., and Walsh, T. R. (1999) Structure and function of metallo-â-lactamases, Recent Res. DeV. Antimicrob. Agents Chemother. 3, 105-132. 15. Carfi, A., Duee, E., Galleni, M., Fre`re, J. M., and Dideberg, O. (1998) 1.85 Å resolution structure of the zinc (II) â-lactamase from Bacillus cereus, Acta Crystallogr., Sect. D 54, 313-323. 16. Carfi, A., Duee, E., Paul-Soto, R., Galleni, M., Fre`re, J. M., and Dideberg, O. (1998) X-ray structure of the Zn(II) â-lactamase from Bacteroides fragilis in an orthorhombic crystal form, Acta Crystallogr., Sect. D 54, 45-57. 17. Concha, N. O., Rasmussen, B. A., Bush, K., and Herzberg, O. (1997) Crystal structure of the cadmium- and mercury-substituted metallo-â-lactamase from Bacteroides fragilis, Protein Sci. 6, 2671-2676. 18. Paul-Soto, R., Zeppezauer, M., Adolph, H. W., Galleni, M., Fre`re, J. M., Carfi, A., Dideberg, O., Wouter, J., Hemmingsen, L., and Bauer, R. (1999) Preference of Cd(II) and Zn(II) for the two metal sites in Bacillus cereus â-lactamase II: a perturbed angular correlation of ç-rays (PAC) spectroscopy study, Biochemistry 38, 16500-16506. 19. de Seny, D, Heinz, U., Wommer, S., Kiefer, M., Meyer-Klaucke, W., Galleni, M., Fre`re, J. M., Bauer, R., and Adolph, H. W. (2001) Metal ion binding and coordination geometry for wild type and mutants of metallo-â-lactamase from Bacillus cereus 569/H/9 (BcII); a combined thermodynamic, kinetic and spectroscopic approach, J. Biol. Chem. 276, 45065-45078. 20. Wommer, S., Rival, S., Heinz, U., Galleni, M., Fre`re, J. M., Franceschini, N., Amicosante, G., Rasmussen, B., Bauer, R., and Adolph, H. W. (2002) Substrate activated zinc binding of metallo- â-lactamases; physiological importance of the mononuclear enzymes, J. Biol. Chem. 277, 24142-24147. 21. Crowder, M. W., Wang, Z., Franklin, S. L., Zovinka, E. P., and Benkovic, S. J. (1996) Characterization of the metal-binding sites of the â-lactamase from Bacteroides fragilis, Biochemistry 35, 12126-12132. 22. Fast, W., Wang, Z., and Benkovic, S. J. (2001) Familial mutations and zinc stoichiometry determine the rate-limiting step of nitrocefin hydrolysis by metallo-â-lactamase from Bacteroides fragilis, Biochemistry 40, 1640-1650. 23. Mitic, N., Smith, S. J., Neves, A., Guddat, L. W., Gahan, L. R., and Schenk, G. (2006) The catalytic mechanisms of binuclear metallohydrolases, Chem. ReV., in press. Weston, J. (2005) Mode of action of bi- and trinuclear zinc hydrolases and their synthetic analogues, Chem. ReV. 105, 2151-2174. Enzyme Deactivation Due to Metal-Ion Dissociation Biochemistry, Vol. 45, No. 36, 2006 11019 24. Auld, D. S. (1995) Removal and replacement of metal ions in metallopepatidases, Methods Enzymol. 248, 228-242. Maret, W., and Vallee, B. L. (1993) Cobalt as probe and label of proteins, Methods Enzymol. 226, 52-71. Vila, A. J., and Fernandez, C. O. (1997) Alkaline transition of Rhus Vernicifera stellacyanin, an unusual blue copper protein, Biochemistry 36, 10566-10570. Guo, J. Q., Wang, S. K., Dong, J., Qiu, H. W., Scott, R. A., and Giedroc, D. P. (1995) X-ray and visible absorption spectroscopy of wildtype and mutant T4 gene 32 proteins: His61, not His81 is the nonthiolate zinc ligand, J. Am. Chem. Soc. 117, 9437-9440. 25. Bertini, I., Johnsson, B. H., Luchinat, C., Pierattelli, R., and Vila, A. J. (1994) Strategies of signal assignments in paramagnetic metalloproteins. An NMR investigation of the thiocyanate adduct of the cobalt (II) substituted human carbonic anhydrase II, J. Magn. Reson., Ser. B 104, 230-239. Oz, G., Pountney, D. L., and Armitage, I. M. (1998) NMR spectroscopic studies of I)1/2 metal ions in biological systems, Biochem. Cell Biol. 76, 223- 234. Bennet, B., and Holz, R. C. (1997) EPR studies on the monoand dicobalt(II)-substituted forms of the aminopeptidase from Aeromonas proteolytica. Insight into the catalytic mechanism of dinuclear hydrolases, J. Am. Chem. Soc. 119, 1923-1933. 26. Bauer, R., Adolph, H. W., Andersson, I., Danielsen, E., Formicka, G., and Zeppezauer, M. (1991) Coordination geometry for cadmium in the catalytic zinc site of horse liver alcohol dehydrogenase: studies by PAC spectroscopy, Eur. Biophys. J. 20, 215-221. 27. Bicknell, R., Knott-Hunziker, Y., and Waley, S. G. (1983) The pH-dependence of class B and class C â-lactamases, Biochem. J. 213, 61-66. 28. Baldwin, G. S., Edwards, G. F. StL., Kiener, P. A., Tully, M. J., Waley, S. G., and Abraham, E. P. (1980) Production of a variant of beta-lactamase II with selectively decreased cephalosporinase activity by a mutant of Bacillus cereus 569/H/9, Biochem. J. 191, 111-116. 29. Hemmingsen, L., Damblon, C., Antony, J., Jensen, M., Adolph, H. W., Wommer, S., Roberts, G. C. K., and Bauer, R. (2001) Dynamics of mononuclear cadmium beta-lactamase revealed by the combination of NMR and PAC spectroscopy, J. Am. Chem. Soc. 123, 10329-10335. 30. Damblon, C., Jensen, M., Ababou, A., Barsukov, I., Papamicael, C., Schofield, C. J., Olsen, L., Bauer, R., and Roberts, G. C. (2003) The inhibitor thiomandelic acid binds to both metal ions in metallo-beta-lactamase and induces positive cooperativity in metal binding, J. Biol. Chem. 31, 29240-29251. 31. Wang, Z., and Benkovic, S. J. (1998) Purification, characterization, and kinetic studies of a soluble Bacteroides fragilis metallo-â- lactamase that provides multiple antibiotic resistance, J. Biol. Chem. 273, 22402-22408. 32. Myers, J. L., and Shaw, R. W. (1989) Production, purification and spectral properties of metal-dependent â-lactamase from Bacillus cereus, Biochim. Biophys. Acta 995, 264-272. 33. Bicknell, R., and Waley, S. G. (1985) Cryoenzymology of Bacillus cereus â-lactamase II, Biochemistry 24, 6876-6887. 34. Bicknell, R., Schaffer, A, Waley, S. G., and Auld, D. S. (1986) Changes in the coordination geometry of the active-site metal during catalysis of benzylpenicillin hydrolysis by Bacillus cereus â-lactamase II, Biochemistry 25, 7208-7215. 35. Bounaga, S., Laws, A. P., Galleni, M., and Page, M. I. (1998) The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent â-lactamase, Biochem J. 331, 703-711. 36. Rasia, R. M., and Vila, A. J. (2004) Structural determinants of substrate binding to Bacillus cereus metallo-â-lactamase, J. Biol. Chem. 279, 26046-26051. 37. Schowen, K. B., and Schowen, R. L. (1982) Solvent isotope effects on enzyme systems, Methods Enzymol. 87, 551-606. 38. Badarau, A. (2006) Reactivity and Inhibition of Metallo-â- lactamases, Ph.D. Thesis, University of Huddersfield, Huddersfield, U.K.
Depositing User: Briony Heyhoe
Date Deposited: 16 Oct 2007
Last Modified: 28 Aug 2021 10:37


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