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.
Hart
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Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
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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
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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