Identification of Asp-130 as the catalytic nucleophile in the main alpha-galactosidase from Phanerochaete chrysosporium, a family 27 glycosyl hydrolase.
Characterization of the complete gene sequence encoding the alpha-galactosidase from Phanerochaete chrysosporium confirms that this enzyme is a member of glycosyl hydrolase family 27 [Henrissat, B., and Bairoch, A. (1996) Biochem. J. 316, 695-696]. This family, together with the family 36 alpha-galactosidases, forms glycosyl hydrolase clan GH-D, a superfamily of alpha-galactosidases, alpha-N-acetylgalactosaminidases, and isomaltodextranases which are likely to share a common catalytic mechanism and structural topology. Identification of the active site catalytic nucleophile was achieved by labeling with the mechanism-based inactivator 2',4', 6'-trinitrophenyl 2-deoxy-2,2-difluoro-alpha-D-lyxo-hexopyranoside; this inactivator was synthesized by anomeric deprotection of the known 1,3,4,6-tetra-O-acetyl-2-deoxy-2, 2-difluoro-D-lyxo-hexopyranoside [McCarter, J. D., Adam, M. J., Braun, C., Namchuk, M., Tull, D., and Withers, S. G. (1993) Carbohydr. Res. 249, 77-90], picrylation with picryl fluoride and 2, 6-di-tert-butylpyridine, and O-deacetylation with methanolic HCl. Enzyme inactivation is a result of the formation of a stable 2-deoxy-2,2-difluoro-beta-D-lyxo-hexopyranosyl-enzyme intermediate. Following peptic digestion, comparative liquid chromatographic/mass spectrometric analysis of inactivated and control enzyme samples served to identify the covalently modified peptide. After purification of the labeled peptide, benzylamine was shown to successfully replace the 2-deoxy-2,2-difluoro-D-lyxo-hexopyranosyl peptidyl ester by aminolysis. The labeled amino acid was identified as Asp-130 of the mature protein by further tandem mass spectrometric analysis of the native and derivatized peptides in combination with Edman degradation analysis. Asp-130 is found within the sequence YLKYDNC, which is highly conserved in all known family 27 glycosyl hydrolases.
Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
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Hart
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Chany
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Withers
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Sims
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Sinnott
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Brumer
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D005800:Genes, Fungal; D006026:Glycoside Hydrolases / Q000145:classification*; D006027:Glycosides / Q000138:chemical synthesis / Q000378:metabolism; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009574:Nitro Compounds / Q000138:chemical synthesis / Q000378:metabolism; D020075:Phanerochaete / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D000519:alpha-Galactosidase / Q000037:antagonists & inhibitors / Q000145:classification / Q000235:genetics / Q000378:metabolism*
C412703:2',4',6',-trinitrophenyl-2-deoxy-2,2-difluoro-alpha-glycoside; D006027:Glycosides; D007202:Indicators and Reagents; D009574:Nitro Compounds; D006026:Glycoside Hydrolases; D000519:alpha-Galactosidase