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Mechanistic Insights from Substrate Preference in Unsaturated Glucuronyl Hydrolase
Author(s) -
Jongkees Seino A. K.,
Yoo Hayoung,
Withers Stephen G.
Publication year - 2014
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201300547
Subject(s) - chemistry , hydrolase , stereochemistry , disaccharide , substrate (aquarium) , moiety , glycoside hydrolase , enzyme , aglycone , transition state analog , active site , glycoside , organic chemistry , oceanography , geology
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 n M −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.