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Mass Spectrometry of Proteins and Peptides pp 203–222Cite as

Identification of Active Site Residues in Glycosidases by Use of Tandem Mass Spectrometry

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 146))

Abstract

The glycosidases are a class of enzymes that are responsible for the hydrolysis of glycosidic bonds. Such glycosidic linkages occur in a wide range of contexts, including polysaccharides, oligosaccharides, glycolipids, glycoproteins, lipopolysaccharides, proteoglycans, saponins, and a range of other glycoconju-gates. Corresponding to this diverse collection of substrates there is a very large assortment of glycosidases responsible for their selective hydrolysis. Amino acid sequences are now available for well over 2000 of these enzymes, and these have been arranged into families on the basis of sequence similarities (1–4). At the last count (November 1999) there were 76 such families, and a regularly updated list of these is readily available at the URL http://afmb.cnrs-mrs.fr/ R~pedro/CAZY/db.html. A large amount of effort has been expended on structural studies of these enzymes in the past 10 yr with the result that three-dimensional X-ray crystal structures are now available for representatives of at least 27 of these families (4–6). These structures are remarkably diverse, with monomer sizes ranging from approx 14,000 to 170,000 Daltons, and compositions ranging from essentially completely α-helical to almost exclusively β-sheet. The reasons for this diversity are probably twofold; the diverse nature of the substrates themselves, and different evolutionary pathways to the construction of an active site.

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References

  1. Henrissat B. (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 280, 309–316.

    PubMed  CAS  Google Scholar 

  2. Henrissat B. and Bairoch A. (1993) New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 293, 781–788.

    PubMed  CAS  Google Scholar 

  3. Henrissat B. and Bairoch A. (1996) Updating the sequence-based classification of glycosyl hydrolases. Biochem. J. 316, 695–696.

    PubMed  Google Scholar 

  4. Henrissat B. and Davies G. (1997) Structural and sequence-based classification of glycoside hydrolases. Curr. Opin. Struct. Biol. 7, 637–644.

    Article  PubMed  CAS  Google Scholar 

  5. Davies G. and Henrissat B. (1995) Structures and mechanisms of glycosyl hydrolases. Structure 3, 853–859.

    Article  PubMed  CAS  Google Scholar 

  6. Davies G., Sinnott M. L., and Withers S. G. (1998) Glycosyl transfer, in Comprehensive Biological Catalysis, vol. 1 (Sinnott M. L., ed.), Academic, New York, pp. 119–208.

    Google Scholar 

  7. Legler G. (1990) Glycoside hydrolases: mechanistic information from studies with reversible and irreversible inhibitors. Adv. Carb. Chem. Biochem. 48, 319–385.

    Article  CAS  Google Scholar 

  8. McCarter J. D. and Withers S. G. (1994) Mechanisms of enzymatic glycoside hydrolysis. Curr. Opin. Struct. Biol. 4, 885–892.

    Article  PubMed  CAS  Google Scholar 

  9. Withers S. G. and Aebersold R. (1995) Approaches to labeling and identification of active site residues in glycosidases. Protein Sci. 4, 361–372.

    Article  PubMed  CAS  Google Scholar 

  10. Sinnott M. L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171–1202.

    Article  CAS  Google Scholar 

  11. Mackenzie L. F., Wang Q., Warren R. A. J., and Withers S. G. (1998) Glycosynthases: mutant glycosidases for oligosaccharide synthesis. J. Am. Chem. Soc. 120, 5583–5584.

    Article  CAS  Google Scholar 

  12. Gebler J. C., Trimbur D. E., Warren R. A. J., Aebersold R., Namchuk M., and Withers S. G. (1995) Substrate-induced inactivation of a crippled β-glucosidase mutant: identification of the labeled amino acid and mutagenic analysis of its role. Biochemistry 34, 14,547–14,533.

    Article  PubMed  CAS  Google Scholar 

  13. Lawson S. L., Wakarchuk W. W., and Withers S. G. (1997) Positioning the acid/base catalyst in a glycosidase: studies with Bacillus circulans xylanase. Biochemistry, 36, 2257–2265.

    Article  PubMed  CAS  Google Scholar 

  14. MacLeod A. M., Lindhorst T., Withers S. G., and Warren R. A. J. (1994) The acid/base catalyst in the exoglucanase/xylanase from Cellulomonas fimi is Glu127: evidence from detailed Kinetic studies of mutants. Biochemistry 33, 6371–6376.

    Article  PubMed  CAS  Google Scholar 

  15. MacLeod A. M., Tull D., Rupitz K., Warren R. A. J., and Withers S. G. (1996) Mechanistic consequences of mutation of active site carboxylates in a retaining β-1,4-glycanase from Cellulomonas fimi. Biochemistry 35, 13,165–13,172.

    Article  PubMed  CAS  Google Scholar 

  16. Mosi R., He S., Uitdehaag J., Dijkstra B. W., and Withers S. G. (1997) Trapping and characterization of the reaction intermediate in cyclodextrin glycosyltransferase by use of activated substrates and a mutant enzyme. Biochemistry 36, 9927–9934.

    Article  PubMed  CAS  Google Scholar 

  17. Notenboom V., Birsan C., Nitz M., Rose D. R., Warren R. A. J., and Withers S. G. (1998) Active site mutations lead to covalent intermediate accumulation in Cellulomonas fimi β-1,4-glycosidase Cex: insights into transition state stabilization. Nature Struct. Biol. 5, 812–818.

    Article  PubMed  CAS  Google Scholar 

  18. Wang Q., Trimbur D., Graham R., Warren R. A. J., and Withers S. G. (1995) Identification of the acid/base catalyst in Agrobacterium faecalis β-glucosidase by Kinetic analysis of mutants. Biochemistry 34, 14,554–14,562.

    Article  PubMed  CAS  Google Scholar 

  19. Withers S. G. (1995) Probing of glycosidase active sites through labeling, mutagenesis and Kinetic studies, in Carbohydrate Bio engineering (Petersen S. B., Svensson B., and Pedersen S., eds.), Elsinore, Denmark, pp. 97–111.

    Google Scholar 

  20. Braun C., Lindhorst T., Madsen N. B., and Withers S. G. (1996) Identification of Asp 549 as the catalytic nucleophile of glycogen-debranching enzyme via trapping of the glycosyl-enzyme intermediate. Biochemistry 35, 5458–5463.

    Article  PubMed  CAS  Google Scholar 

  21. Sinnott M. L. and Souchard I. J. L. (1973) The mechanism of action of β-galactosidase: effect of aglycone nature and α-deuterium substitution on the hydrolysis of aryl galactosides. Biochem. J. 133, 89–98.

    PubMed  CAS  Google Scholar 

  22. Kempton J. B. and Withers S. G. (1992) Mechanism of Agrobacterium faecalis β-glucosidase: Kinetic studies. Biochemistry 31, 9961–9969.

    Article  PubMed  CAS  Google Scholar 

  23. Tull D. and Withers S. G. (1994) Mechanisms of cellulases and xylanases: a detailed Kinetic study of the exo-β-1,4-glycanase from Cellulomonas fimi. Biochemistry 33, 6363–6370.

    Article  PubMed  CAS  Google Scholar 

  24. Withers S. G., Street I. P., Bird P., and Dolphin D. H. (1987) 2-Deoxy-2-fluoro-glucosides: a novel class of mechanism-based glucosidase inhibitors. J. Am. Chem. Soc. 109, 7530–7531.

    Article  CAS  Google Scholar 

  25. Withers S. G., Rupitz K., and Street I. P. (1988) 2-Deoxy-2-fluoro-D-glycosyl fluorides. A new class of specific mechanism-based glycosidase inhibitors. J. Biol. Chem. 263, 7929–7932.

    PubMed  CAS  Google Scholar 

  26. Wolfenden R. and Kati W. M. (1991) Testing the limits of protein-ligand binding discrimination with transition-state analogue inhibitors. Acc. Chem. Res. 24, 209–215.

    Article  CAS  Google Scholar 

  27. Wentworth D. F. and Wolfenden R. (1974) Slow binding of D-galactal, a “reversible” inhibitor of bacterial β-galactosidase. Biochemistry 13, 4715–4720.

    Article  PubMed  CAS  Google Scholar 

  28. Roeser K. R. and Legler G. (1981) Role of sugar hydroxyl groups in glycoside hydrolysis. Cleavage mechanism of deoxyglucosidases and related substrates by β-glucosidase A3 from Aspergillus wentii. Biochim. Biophys. Acta 657, 321–333.

    Article  PubMed  CAS  Google Scholar 

  29. Namchuk M. N. and Withers S. G. (1995) Mechanism of Agrobacterium β-glucosidase: Kinetic analysis of the role of noncovalent enzyme/substrate interactions. Biochemistry 34, 16,194–16,202.

    Article  PubMed  CAS  Google Scholar 

  30. McCarter J. D., Adam M. J., and Withers S. G. (1992) Binding energy and catalysis. Fluorinated and deoxygenated glycosides as mechanistic probes of Escherichia coli (lacZ) β-galactosidase. Biochem. J. 286, 721–727.

    PubMed  CAS  Google Scholar 

  31. Street I. P., Rupitz K., and Withers S. G. (1989) Fluorinated and deoxygenated substrates as probes of transition-state structure in glycogen phosphorylase. Biochemistry 28, 1581–1587.

    Article  PubMed  CAS  Google Scholar 

  32. White A., Tull D., Johns K., Withers S. G., and Rose D. R. (1996) Crystallo-graphic observation of a covalent catalytic intermediate in a β-glycosidase. Nature Struct. Biol. 3, 149–154.

    Article  PubMed  CAS  Google Scholar 

  33. Street I. P., Kempton J. B., and Withers S. G. (1992) Inactivation of a β-gluco-sidase through the accumulation of a stable 2-deoxy-2-fluoro-α-D-glucopyranosyl-enzyme intermediate: a detailed investigation. Biochemistry 31, 9970–9978.

    Article  PubMed  CAS  Google Scholar 

  34. Withers S. G. and Street I. P. (1988) Identification of a covalent α-D-gluco-pyranosyl enzyme intermediate formed on a β-glucosidase. J. Am. Chem. Soc. 110, 8551–8553.

    Article  CAS  Google Scholar 

  35. McCarter J. D., Adam M. J., Braun C., Namchuk M., Tull D., and Withers S. G. (1993) Syntheses of 2-deoxy-2-fluoro mono-and oligo-saccharide glycosides from glycals and evaluation as glycosidase inhibitors. Carbohydr. Res. 249, 77–90.

    Article  PubMed  CAS  Google Scholar 

  36. Howard S., He S., and Withers S. G. (1998) Identification of the active site nucleophile in jack bean α-mannosidase using 5-fluoro-β-l-gulosyl fluoride. J. Biol. Chem. 273, 2067–2072.

    Article  PubMed  CAS  Google Scholar 

  37. McCarter J. D. and Withers S. G. (1996) Unequivocal identification of Asp-214 as the catalytic nucleophile of Saccharomyces cerevisiae β-glucosidase using 5-fluoro glycosyl fluorides. J. Biol. Chem. 271, 6889–6894.

    Article  PubMed  CAS  Google Scholar 

  38. McCarter J. D. and Withers S. G. (1996) 5-Fluoro glycosides: a new class of mechanism-based inhibitors of both α-and β-glucosidases. J. Am. Chem. Soc. 118, 241–242.

    Article  CAS  Google Scholar 

  39. Braun C., Brayer G. D., and Withers S. G. (1995) Mechanism-based inhibition of yeast α-glucosidase and human pancreatic α-amylase by a new class of inhibitors. 2-Deoxy-2,2-difluoro-(-glycosides. J. Biol. Chem. 270, 26,778–26,781.

    Article  PubMed  CAS  Google Scholar 

  40. Withers S. G., Warren R. A. J., Street I. P., Rupitz K., Kempton J. B., and Aebersold R. (1990) Unequivocal demonstration of the involvement of a glutamate residue as a nucleophile in the mechanism of a ‘retaining’ glycosidase. J. Am. Chem. Soc. 112, 5887–5889.

    Article  CAS  Google Scholar 

  41. Gebler J. C., Aebersold R., and Withers S. G. (1992) Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) β-galactosidase from Escherichia coli. J. Biol. Chem. 267, 11,126–11,130.

    PubMed  CAS  Google Scholar 

  42. Tull D., Withers S. G., Gilkes N. R., Kilburn D. G., Warren R. A., and Aebersold R. (1991) Glutamic acid 274 is the nucleophile in the active site of a “retaining” exoglucanase from Cellulomonas fimi. J. Biol. Chem. 266, 15,621–15,625.

    PubMed  CAS  Google Scholar 

  43. Tull D., Miao S., Withers S. G., and Aebersold R. (1994) Identification of derivatised peptides without radiolabels: tandem mass spectrometric localisation of the tagged active site nucleophiles of two cellulases and a β-glucosidase. Anal. Biochem. 224, 509–514.

    Article  Google Scholar 

  44. Legler G. and Hasnain S. N. (1970) Markierung des aktiven Zentrums der β-Glucosidasen A und B aus dem Suβmandel-emulsin mit [3H]6-Brom-6-desoxy-condurit-B-epoxid. Hoppe-Seylers Z. Physiol. Chem. 351, 25–31.

    Article  PubMed  CAS  Google Scholar 

  45. Legler G. (1968) Untersuchungen zum Wirkungsmechanismus glykosidspal-tender Enzyme, III. Markierung des aktiven Aentrums einer β-Glucosidase aus Aspergillus wentii mit [14C]Condrurit-B-epoxid. Hoppe-Seyler’s Z. Physiol. Chem. 349, 767–774.

    Article  PubMed  CAS  Google Scholar 

  46. Miao S., Ziser L., Aebersold R., and Withers S. G. (1994) Identification of glutamic acid 78 as the active site nucleophile in Bacillus subtilis xylanase using electrospray tandem mass spectrometry. Biochemistry 33, 7027–7032.

    Article  PubMed  CAS  Google Scholar 

  47. Wong A. W., He S., Grubb J. H., Sly W. S., and Withers S. G. (1998) Identification of Glu540 as the catalytic nucleophile of human β-glucuronidase using electrospray mass spectrometry. J. Biol. Chem. 273, 34,057–34,062.

    Article  PubMed  CAS  Google Scholar 

  48. He S. and Withers S. G. (1997) Assignment of sweet almond β-glucosidase as a family 1 glycosidase and identification of its active site nucleophile. J. Biol. Chem. 272, 24,864–24,867.

    Article  PubMed  CAS  Google Scholar 

  49. Mackenzie L. F., Brooke G. S., Cutfield J. F., Sullivan P. A., and Withers S. G. (1997) Identification of glu-330 as the catalytic nucleophile of Candida albicans exo-β-(1,3)-glucanase. J. Biol. Chem. 272, 3161–3167.

    Article  PubMed  CAS  Google Scholar 

  50. Mackenzie L. F., Davies G. J., Schulein M., and Withers S. G. (1997) Identification of the catalytic nucleophile of endoglucanase I from Fusarium oxysporum by mass spectrometry. Biochemistry 36, 5893–5901.

    Article  PubMed  CAS  Google Scholar 

  51. Mackenzie L. F., Sulzenbacher G., Divne C., Jones T. A., Woldike H. F., Schulein M., et al. J. (1998) Crystal structure of the family 7 endoglucanase I (CelB) from Humicola insolens at 2.2 A resolution and identification of the catalytic nucleophile by trapping of the covalent glycosyl-enzyme intermediate. Biochem. J. 335, 409–416.

    PubMed  CAS  Google Scholar 

  52. McCarter J. D., Burgoyne D. L., Miao S. C., Zhang S. Q., Callahan J. W., and Withers S. G. (1997) Identification of glu-268 as the catalytic nucleophile of human lysosomal β-galactosidase precursor by mass spectrometry. J. Biol. Chem. 272, 396–400.

    Article  PubMed  CAS  Google Scholar 

  53. Miao S., McCarter J. D., Grace M. E., Grabowski G. A., Aebersold R., and Withers S. G. (1994) Identification of Glu340 as the active-site nucleophile in human glucocerebrosidase by use of electrospray tandem mass spectrometry. J. Biol. Chem. 269, 10,975–10,978.

    PubMed  CAS  Google Scholar 

  54. Vocadlo D. J., Mackenzie L. F., He S., Zeikus G., and Withers S. G. (1998) Identification of Glu277 as the catalytic nucleophile of Thermoanaerobacterium saccharolyticum β-xylosidase using electrospray MS. Biochem. J. 335, 449–455.

    PubMed  CAS  Google Scholar 

  55. Zechel D. L., He S., Dupont C., and Withers S. G. (1998) Identification of Glu 120 as the catalytic nucleophile in Streptomyces lividans endoglucanase CelB. Biochem. J. 336, 139–145.

    PubMed  CAS  Google Scholar 

  56. Lee Y.-E. and Zeikus J. G. (1993) Genetic organization, sequence and biochemical characterization of recombinant beta-xylosidase from Thermoanerobacterium saccharolyticum strain B6A-R1. J. Gen. Microbiol. 139, 1235–1243.

    PubMed  CAS  Google Scholar 

  57. Ziser L., Setyawati I., and Withers S. G. (1995) Syntheses and testing of substrates and mechanism-based inactivators for xylanases. Carbohydr. Res. 274, 137–153.

    Article  PubMed  CAS  Google Scholar 

  58. Leatherbarrow, R. J. (1992) Gra-Fit, Version 2.0, Erithacus Software, Staines, UK.

    Google Scholar 

  59. Armand S., Vielle C., Gey C., Heyraud A., Zeikus J. G., and Henrissat B. (1996) Stereochemical course and reaction products of the action of β-xylosidase from Thermoanaerobacterium saccharolyticum strain B6A-RI. Eur. J. Biochem. 263, 706–713.

    Article  Google Scholar 

  60. Henrissat B., Callebaut I., Fabrega S., Lehn P., Mornon J. P., and Davies G. (1995) Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases. Proc. Natl. Acad. Sci. USA 92, 7090–7094.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Chemistry, University of British Columbia, Vancouver, Canada

    David J. Vocadlo & Stephen G. Withers

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  1. David J. Vocadlo
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  1. Sale, Manchester, UK

    John R. Chapman

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Vocadlo, D.J., Withers, S.G. (2000). Identification of Active Site Residues in Glycosidases by Use of Tandem Mass Spectrometry. In: Chapman, J.R. (eds) Mass Spectrometry of Proteins and Peptides. Methods in Molecular Biology™, vol 146. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-045-4:203

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  • DOI: https://doi.org/10.1385/1-59259-045-4:203

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-0-89603-609-3

  • Online ISBN: 978-1-59259-045-2

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