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. 2000 Nov 1;351(Pt 3):833–838.

Identification of Glu-519 as the catalytic nucleophile in beta-mannosidase 2A from Cellulomonas fimi.

D Stoll 1, S He 1, S G Withers 1, R A Warren 1
PMCID: PMC1221426  PMID: 11042141

Abstract

Incubation of the beta-mannosidase Man2A from Cellulomonas fimi with 2-deoxy-2-fluoro-beta-D-mannosyl fluoride (2FMan beta F) resulted in time-dependent inactivation of the enzyme (inactivation rate constant k(i)=0.57 min(-1), dissociation constant for the inactivator K(i)=0.41 mM) through the accumulation of a covalent 2-deoxy-2-fluoro-alpha-D-mannosyl-beta-mannosidase 2A (2FMan-Man2A) enzyme intermediate, as observed by electrospray ionization mass spectrometry. The stoichiometry of inactivation was 1:1. Removal of excess inactivator and regeneration of active enzyme by transglycosylation of the covalently attached inhibitor to gentiobiose [Glc beta(1-6)Glc] demonstrated that the covalent intermediate was catalytically competent. Comparison by MS of the peptic digests of 2FMan-Man2A with peptic digests of native Man2A revealed a peptide of m/z 1520 that was unique to 2FMan-Man2A, and one of m/z 1036.5 that was unique to a Man2A peptide. Their sequences, determined by collision-induced fragmentation, were CSEFGFQGPPTW and FGFQGPPTW, corresponding to residues 517-528 and 520-528 of Man2A respectively. The difference in mass of 483.5 between the two peptides equals the sum of the masses of the tripeptide CSE plus that of 2-fluoromannose. It was concluded that in 2FMan-Man2A, the 2-fluoromannose esterified to Glu-519 blocks hydrolysis of the Glu-519-Phe-520 peptide bond, and that Glu-519 is the catalytic nucleophile in this enzyme. This residue is conserved in all members of family 2 of the glycosyl hydrolases. This represents the first ever labelling and identification of an active-site nucleophile in a beta-mannosidase.

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Selected References

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

  1. Dan S., Marton I., Dekel M., Bravdo B. A., He S., Withers S. G., Shoseyov O. Cloning, expression, characterization, and nucleophile identification of family 3, Aspergillus niger beta-glucosidase. J Biol Chem. 2000 Feb 18;275(7):4973–4980. doi: 10.1074/jbc.275.7.4973. [DOI] [PubMed] [Google Scholar]
  2. Gebler J. C., Aebersold R., Withers S. G. Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) beta-galactosidase from Escherichia coli. J Biol Chem. 1992 Jun 5;267(16):11126–11130. [PubMed] [Google Scholar]
  3. Henrissat B., Bairoch A. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J. 1993 Aug 1;293(Pt 3):781–788. doi: 10.1042/bj2930781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Jacobson R. H., Zhang X. J., DuBose R. F., Matthews B. W. Three-dimensional structure of beta-galactosidase from E. coli. Nature. 1994 Jun 30;369(6483):761–766. doi: 10.1038/369761a0. [DOI] [PubMed] [Google Scholar]
  5. Jain S., Drendel W. B., Chen Z. W., Mathews F. S., Sly W. S., Grubb J. H. Structure of human beta-glucuronidase reveals candidate lysosomal targeting and active-site motifs. Nat Struct Biol. 1996 Apr;3(4):375–381. doi: 10.1038/nsb0496-375. [DOI] [PubMed] [Google Scholar]
  6. Mackenzie L. F., Brooke G. S., Cutfield J. F., Sullivan P. A., Withers S. G. Identification of Glu-330 as the catalytic nucleophile of Candida albicans exo-beta-(1,3)-glucanase. J Biol Chem. 1997 Feb 7;272(6):3161–3167. doi: 10.1074/jbc.272.6.3161. [DOI] [PubMed] [Google Scholar]
  7. Mackenzie L. F., Davies G. J., Schülein M., Withers S. G. Identification of the catalytic nucleophile of endoglucanase I from Fusarium oxysporum by mass spectrometry. Biochemistry. 1997 May 13;36(19):5893–5901. doi: 10.1021/bi962962h. [DOI] [PubMed] [Google Scholar]
  8. McCarter J. D., Adam M. J., Withers S. G. Binding energy and catalysis. Fluorinated and deoxygenated glycosides as mechanistic probes of Escherichia coli (lacZ) beta-galactosidase. Biochem J. 1992 Sep 15;286(Pt 3):721–727. doi: 10.1042/bj2860721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. McCarter J. D., Burgoyne D. L., Miao S., Zhang S., Callahan J. W., Withers S. G. Identification of Glu-268 as the catalytic nucleophile of human lysosomal beta-galactosidase precursor by mass spectrometry. J Biol Chem. 1997 Jan 3;272(1):396–400. doi: 10.1074/jbc.272.1.396. [DOI] [PubMed] [Google Scholar]
  10. McCarter J. D., Withers S. G. Unequivocal identification of Asp-214 as the catalytic nucleophile of Saccharomyces cerevisiae alpha-glucosidase using 5-fluoro glycosyl fluorides. J Biol Chem. 1996 Mar 22;271(12):6889–6894. doi: 10.1074/jbc.271.12.6889. [DOI] [PubMed] [Google Scholar]
  11. Miao S., Ziser L., Aebersold R., Withers S. G. Identification of glutamic acid 78 as the active site nucleophile in Bacillus subtilis xylanase using electrospray tandem mass spectrometry. Biochemistry. 1994 Jun 14;33(23):7027–7032. doi: 10.1021/bi00189a002. [DOI] [PubMed] [Google Scholar]
  12. Stoll D., Stålbrand H., Warren R. A. Mannan-degrading enzymes from Cellulomonas fimi. Appl Environ Microbiol. 1999 Jun;65(6):2598–2605. doi: 10.1128/aem.65.6.2598-2605.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Street I. P., Kempton J. B., Withers S. G. Inactivation of a beta-glucosidase through the accumulation of a stable 2-deoxy-2-fluoro-alpha-D-glucopyranosyl-enzyme intermediate: a detailed investigation. Biochemistry. 1992 Oct 20;31(41):9970–9978. doi: 10.1021/bi00156a016. [DOI] [PubMed] [Google Scholar]
  14. Vocadlo D. J., MacKenzie L. F., He S., Zeikus G. J., Withers S. G. Identification of glu-277 as the catalytic nucleophile of Thermoanaerobacterium saccharolyticum beta-xylosidase using electrospray MS. Biochem J. 1998 Oct 15;335(Pt 2):449–455. doi: 10.1042/bj3350449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Vocadlo D. J., Mayer C., He S., Withers S. G. Mechanism of action and identification of Asp242 as the catalytic nucleophile of Vibrio furnisii N-acetyl-beta-D-glucosaminidase using 2-acetamido-2-deoxy-5-fluoro-alpha-L-idopyranosyl fluoride. Biochemistry. 2000 Jan 11;39(1):117–126. doi: 10.1021/bi991958d. [DOI] [PubMed] [Google Scholar]
  16. Wang Q., Tull D., Meinke A., Gilkes N. R., Warren R. A., Aebersold R., Withers S. G. Glu280 is the nucleophile in the active site of Clostridium thermocellum CelC, a family A endo-beta-1,4-glucanase. J Biol Chem. 1993 Jul 5;268(19):14096–14102. [PubMed] [Google Scholar]
  17. Withers S. G., Aebersold R. Approaches to labeling and identification of active site residues in glycosidases. Protein Sci. 1995 Mar;4(3):361–372. doi: 10.1002/pro.5560040302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Withers S. G., Rupitz K., Street I. P. 2-Deoxy-2-fluoro-D-glycosyl fluorides. A new class of specific mechanism-based glycosidase inhibitors. J Biol Chem. 1988 Jun 15;263(17):7929–7932. [PubMed] [Google Scholar]
  19. Wong A. W., He S., Grubb J. H., Sly W. S., Withers S. G. Identification of Glu-540 as the catalytic nucleophile of human beta-glucuronidase using electrospray mass spectrometry. J Biol Chem. 1998 Dec 18;273(51):34057–34062. doi: 10.1074/jbc.273.51.34057. [DOI] [PubMed] [Google Scholar]
  20. Zechel D. L., He S., Dupont C., Withers S. G. Identification of Glu-120 as the catalytic nucleophile in Streptomyces lividans endoglucanase celB. Biochem J. 1998 Nov 15;336(Pt 1):139–145. doi: 10.1042/bj3360139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Zechel D. L., Withers S. G. Glycosidase mechanisms: anatomy of a finely tuned catalyst. Acc Chem Res. 2000 Jan;33(1):11–18. doi: 10.1021/ar970172+. [DOI] [PubMed] [Google Scholar]

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