Identification of the catalytic nucleophile of the family 29 alpha-L-fucosidase from Thermotoga maritima through trapping of a covalent glycosyl-enzyme intermediate and mutagenesis.

Fucose-containing glycoconjugates are key antigenic determinants in many biological processes. A change in expression levels of the enzymes responsible for tailoring these glycoconjugates has been associated with many pathological conditions and it is therefore surprising that little information is known regarding the mechanism of action of these important catabolic enzymes. Thermotoga maritima, a thermophilic bacterium, produces a wide range of carbohydrate-processing enzymes including a 52-kDa alpha-L-fucosidase that has 38% sequence identity and 56% similarity to human fucosidases. The catalytic nucleophile of this enzyme was identified to be Asp-224 within the peptide sequence 222WNDMGWPEKGKEDL235 using the mechanism-based covalent inactivator 2-deoxy-2-fluoro-alpha-L-fucosyl fluoride. The 10(4)-fold lower activity (kcat/Km) of the site-directed mutant D224A, and the subsequent rescue of activity upon addition of exogenous nucleophiles, conclusively confirms this assignment. This article presents the first direct identification of the catalytic nucleophile of an alpha-L-fucosidase, a key step in the understanding of these important enzymes.