@article { ISI:000088835700023,
	title = {Identification of Asp-130 as the catalytic nucleophile in the main alpha-galactosidase from Phanerochaete chrysosporium, a family 27 glycosyl hydrolase},
	journal = {BIOCHEMISTRY},
	volume = {39},
	number = {32},
	year = {2000},
	month = {AUG 15},
	pages = {9826-9836},
	abstract = {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 dan 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{\textquoteright},4{\textquoteright},6{\textquoteright}-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.},
	issn = {0006-2960},
	doi = {10.1021/bi0008074},
	author = {Hart, DO and He, SM and Chany, CJ and Withers, SG and Sims, PFG and Sinnott, ML and Brumer, H}
}
