IDENTIFICATION OF THE ACTIVE-SITE NUCLEOPHILE IN JACK BEAN ALPHA-MANNOSIDASE USING 5-FLUORO-BETA-L-GULOXYL FLUORIDE

Mannosidases play a key role in the processing of glycoproteins and th us are of considerable pharmaceutical interest and indeed have emerged as targets for the development of anti-cancer therapies, Access to us eful quantities of the mammalian enzymes has not yet been achieved; th erefore, jack bean mannosidase, a readily available enzyme, has become the model system, However, the relevance of this enzyme has not been demonstrated, nor is anything known about the active site structure of this, or any other, mannosidase. Hydrolysis by this enzyme occurs wit h net retention of sugar anomeric configuration; thus, a double displa cement mechanism involving a mannosyl-enzyme intermediate is presumabl y involved, Two new mechanism-based inhibitors, 5-fluoro-alpha-D-manno syl fluoride and 5-fluoro-beta-L-gulosyl fluoride, which function by t he steady state trapping of such an intermediate, have been synthesize d and tested, Both show high affinity for jack bean alpha-mannosidase (K-i' = 71 and 86 mu M, respectively), and the latter has been used to label the active site nucleophile. The labeled peptide present in a p eptic digest of this trapped glycosyl-enzyme intermediate was identifi ed by neutral loss scans on an electrospray ionization triple quadrupo le mass spectrometer, Comparative liquid chromatographic/mass spectrom etric analysis of peptic digests of labeled and unlabeled enzyme sampl es confirmed the unique presence of this peptide of mit 1180.5 in the labeled sample, The label was cleaved from the peptide by treatment wi th ammonia, and the resultant unlabeled peptide was purified and seque nced by Edman degradation, The peptide identified contained only one c andidate for the catalytic nucleophile, an aspartic acid, This residue was contained within the sequence Gly-Trp-Gln-Ile-Asp-Pro-Phe-Gly-His -Ser, which showed excellent sequence similarity with regions in mamma lian lysosomal and Golgi cu mannosidase sequences, These mammalian alp ha-mannosidases belong to family 38 (or class II alpha-mannosidases) i n which the Asp in the above sequence is totally conserved, This findi ng therefore assigns jack bean alpha-mannosidase to family 38, validat ing it as a model for other pharmaceutically interesting enzymes and t hereby identifying the catalytic nucleophile within this family.