Identification of the active site nucleophile in jack bean alpha-mannosidase using 5-fluoro-beta-L-gulosyl fluoride.
|
| Abstract |
Mannosidases play a key role in the processing of glycoproteins and thus are of considerable pharmaceutical interest and indeed have emerged as targets for the development of anti-cancer therapies. Access to useful quantities of the mammalian enzymes has not yet been achieved; therefore, 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 with net retention of sugar anomeric configuration; thus, a double displacement mechanism involving a mannosyl-enzyme intermediate is presumably involved. Two new mechanism-based inhibitors, 5-fluoro-alpha-D-mannosyl fluoride and 5-fluoro-beta-L-gulosyl fluoride, which function by the steady state trapping of such an intermediate, have been synthesized and tested. Both show high affinity for jack bean alpha-mannosidase (Ki' = 71 and 86 microM, respectively), and the latter has been used to label the active site nucleophile. The labeled peptide present in a peptic digest of this trapped glycosyl-enzyme intermediate was identified by neutral loss scans on an electrospray ionization triple quadrupole mass spectrometer. Comparative liquid chromatographic/mass spectrometric analysis of peptic digests of labeled and unlabeled enzyme samples confirmed the unique presence of this peptide of m/z 1180.5 in the labeled sample. The label was cleaved from the peptide by treatment with ammonia, and the resultant unlabeled peptide was purified and sequenced by Edman degradation. The peptide identified contained only one candidate 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 mammalian lysosomal and Golgi alpha-mannosidase sequences. These mammalian alpha-mannosidases belong to family 38 (or class II alpha-mannosidases) in which the Asp in the above sequence is totally conserved. This finding therefore assigns jack bean alpha-mannosidase to family 38, validating it as a model for other pharmaceutically interesting enzymes and thereby identifying the catalytic nucleophile within this family.
|
|---|---|
| Authors | S Howard, S He, S G Withers |
| Journal | The Journal of biological chemistry (J Biol Chem) Vol. 273 Issue 4 Pg. 2067-72 (Jan 23 1998) ISSN: 0021-9258 [Print] United States |
| PMID | 9442045 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't) |
| Chemical References |
|
| Topics |
|
Join CureHunter, for free Research Interface BASIC access!
Realize the full power of the drug-disease research graph!