|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|
|Date Published||JAN 3|
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.