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-galactosi dase from Phanerochaete chrysosporium confirms that this enzyme is a member of glycosyl hydrolase family 27 [Henrissat, B., and Bairoch, A. (1996) Bio chem. J. 316, 695-696]. This family, together with the family 36 alpha-gala ctosidases, forms glycosyl hydrolase dan GH-D, a superfamily of alpha-galac tosidases, alpha-N-acetylgalactosaminidases, and isomaltodextranases which are likely to share a common catalytic mechanism and structural topology. I dentification of the active site catalytic nucleophile was achieved by labe ling 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-di fluoro-D-lyxo-hexopyranoside [McCarter, J. D., Adam, M. J., Braun, C., Namc huk, M., Tull, D., and Withers, S. G. (1993) Carbohydr. Res. 249, 77-90], p icrylation with picryl fluoride and 2,6-di-tert-butylpyridine, and O-deacet ylation with methanolic HCl. Enzyme inactivation is a result of the formati on of a stable 2-deoxy-2,2-difluoro-beta-D-lyxo-hexopyranosyl-enzyme interm ediate. 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 labele d peptide, benzylamine was shown to successfully replace the 2-deoxy-2,2-di fluoro-D-lyxo-hexopyranosyl peptidyl ester by aminolysis. The labeled amino acid was identified as Asp-130 of the mature protein by further tandem mas s spectrometric analysis of the native and derivatized peptides in combinat ion with Edman degradation analysis. Asp-130 is found within the sequence Y LKYDNC, which is highly conserved in all known family 27 glycosyl hydrolase s.