Mechanistic Investigations of Unsaturated Glucuronyl Hydrolase from Clostridium perfringens [Glycobiology and Extracellular Matrices]

February 26th, 2014 by Jongkees, S. A. K., You, H., Withers, S. G.

Experiments were carried out to probe the details of the hydration-initiated hydrolysis catalysed by Clostridium perfringens unsaturated glucuronyl hydrolase of glycoside hydrolase family 88 in the CAZy classification system. Direct 1H-NMR monitoring of the enzymatic reaction detected no accumulated reaction intermediates in solution, suggesting that rearrangement of the initial hydration product occurs on-enzyme. An attempt at mechanism-based trapping of on-enzyme intermediates using a 1,1-difluoro-substrate was unsuccessful because the probe was too deactivated to be turned over by the enzyme. Kinetic isotope effects arising from deuterium-for-hydrogen substitution at carbons 1 and 4 provide evidence for separate first-irreversible and overall rate-determining steps in the hydration reaction, with two potential mechanisms proposed to explain these results. Based on the positioning of catalytic residues in the enzyme active site, the lack of efficient turnover of a 2-deoxy-2-fluoro substrate, and several unsuccessful attempts at confirmation of a simpler mechanism involving a covalent glycosyl-enzyme intermediate, the most plausible mechanism is one involving an intermediate bearing an epoxide on carbons 1 and 2.