Conformationally Controlled Reactivity of Carbasugars Uncovers the Choreography of Glycoside Hydrolase Catalysis

摘要

Glycoside hydrolases (GHs) catalyze hydrolyses of glycoconjugates in which the enzyme choreographs a series of conformational changes during the catalytic cycle. As a result, some GH families, including alpha-amylases (GH13), have their chemical steps concealed kinetically. To address this issue for a GH13 enzyme, we prepared seven cyclohexenyl-based carbasugars of alpha-D-glucopyranoside that we show are good covalent inhibitors of a GH13 yeast alpha-glucosidase. The linear free energy relationships between rate constants and pK(a) of the leaving group are curved upward, which is indicative of a change in mechanism, with the better leaving groups reacting by an S(N)1 mechanism, while reaction rates for the worse leaving groups are limited by a conformational change of the Michaelis complex prior to a rapid S(N)2 reaction with the enzymatic nucleophile. Five bicyclo[4.1.0]heptyl-based carbaglucoses were tested with this enzyme, and our results are consistent with pseudoglycosidic bond cleavage that occurs via S(N)1 transition states that include nonproductive binding of the leaving group to the enzyme. In total, we show that the conformationally orthogonal reactions of these two carbasugars reveal mechanistic details hidden by conformational changes that the Michaelis complex of the enzyme and natural substrate undergoes which align the nucleophile for efficient catalysis.