Mechanistic insights from substrate preference in unsaturated glucuronyl hydrolase.
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 kcat =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 kcat /Km =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.
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia, V6T 1Z1 (Canada).
Seino A K
SA
Jongkees
Hayoung
H
Yoo
Stephen G
SG
Withers
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003016:Clostridium perfringens / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D020719:Glucuronides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D013379:Substrate Specificity; D013816:Thermodynamics
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D020719:Glucuronides; D006026:Glycoside Hydrolases; C120983:unsaturated glucuronyl hydrolase
carbohydrates; enzymes; hydrolases; inhibitors; reaction mechanisms