Glycoside Hydrolase Catalysis: Do Substrates and Mechanism-Based Covalent Inhibitors React via Matching Transition States?

In this study, we look at how a catalytically efficient α-galactosidase stabilizes transition state (TS) charge delocalization for substrate hydrolysis. We then assess whether covalent inhibition of the enzyme by three types of mechanism-based covalent inhibitors occurs via similar modes of TS stabilization. We show, using Bartlett-type linear free energy relationships, that good correlations are obtained between the catalytic efficiencies (kcat/Km and/or kinact/Ki) for enzyme-catalyzed reactions of natural and activated galactoside substrates and of representatives of three families of classical mechanism-based inhibitors: a 2-deoxy-2-fluoroglycoside, allylic carbasugars, and an epoxy carbasugar. Of note, we show that glycoside natural substrates and allylic carbasugars display log(rate)–log(rate) correlations that are unity (slope ≈ 1), an observation consistent with them having identical positive charge stabilization at the SN1-like glycosylation and pseudo-glycosylation TSs, respectively. In contrast, 2-deoxy-2-fluoroglycoside mechanism-based inhibitors react via a different enzyme-catalyzed mechanism (SN2), while the strained epoxy carbasugar inactivates the α-galactosidase by traversing a TS in which the glycoside hydrolase stabilizes the inactivation TS that has a significantly lower degree of charge stabilization to those for the natural glycoside substrates. To add weight to these conclusions, we computed free energy landscapes and their associated galactosylation and pseudo-galactosylation TSs using QM/MM molecular dynamics methods with the whole solvated enzyme.