Title | Mechanistic Insights from Substrate Preference in Unsaturated Glucuronyl Hydrolase |
Publication Type | Journal Article |
Year of Publication | 2014 |
Authors | Jongkees, SAK, Yoo, H, Withers, SG |
Journal | CHEMBIOCHEM |
Volume | 15 |
Pagination | 124-134 |
Date Published | JAN 3 |
ISSN | 1439-4227 |
Abstract | Natural and synthetic unsaturated glucuronides were tested as substrates for Clostridium perfringens unsaturated glucuronyl hydrolase to probe its mechanism and to guide inhibitor design. Of the natural substrates, a chondroitin disaccharide substrate with sulfation of the primary alcohol on carbon 6 of its N-acetylgalactosamine moiety was found to have the highest turnover number of any substrate reported for an unsaturated glucuronyl hydrolase, with k(cat)=112 s(-1). Synthetic aryl glycoside substrates with electron-withdrawing aglycone substituents were cleaved more slowly than those with electron-donating substituents. Similarly, an unsaturated glucuronyl fluoride was found to be a particularly poor substrate, with k(cat)/K-m=44 nM(-1)s(-1)a very unusual result for a glycoside-cleaving enzyme. These results are consistent with a transition state with positive charge at carbon 5 and the endocyclic oxygen, as anticipated in the hydration mechanism proposed. However, several analogues designed to take advantage of strong enzyme binding to such a transition state showed little to no inhibition. This result suggests that further work is required to understand the true nature of the transition state stabilised by this enzyme. |
DOI | 10.1002/cbic.201300547 |