Substrate-binding recognition and specificity of trehalose phosphorylase from Schizophyllum commune examined in steady-state kinetic studies with deoxy and deoxyfluoro substrate analogues and inhibitors.

Abstract

Trehalose phosphorylase is a component of the alpha-D-glucopyranosyl alpha-D-glucopyranoside (alpha,alpha-trehalose)-degrading enzyme system in fungi and it catalyses glucosyl transfer from alpha,alpha-trehalose to phosphate with net retention of the anomeric configuration. The enzyme active site has no detectable affinity for alpha,alpha-trehalose in the absence of bound phosphate and catalysis occurs from the ternary complex. To examine the role of non-covalent enzyme-substrate interactions for trehalose phosphorylase recognition, we used the purified enzyme from Schizophyllum commune and tested a series of incompetent structural analogues of the natural substrates and products as inhibitors of the enzyme. Equilibrium-binding constants (K(i)) for deoxy- and deoxyfluoro derivatives of D-glucose show that loss of interactions with the 3-, 4- or 6-OH, but not the reactive 1- and the 2-OH, results in considerably (> or =100-fold) weaker affinity for sugar-binding subsite +1, revealing the requirement for hydrogen bonding with hydroxyls, away from the site of chemical transformation to position precisely the D-glucose-leaving group/nucleophile for catalysis. The high specificity of trehalose phosphorylase for the sugar aglycon during binding and conversion of O-glycosides is in contrast with the observed alpha-retaining phosphorolysis of alpha-D-glucose-1-fluoride (alpha-D-Glc-1-F) since the productive bonding capability of the fluoride-leaving group with subsite +1 is minimal. The specificity constant (19 M(-1).s(-1)) and catalytic-centre activity (0.1 s(-1)) for the reaction with alpha-D-Glc-1-F are 0.10- and 0.008-fold the corresponding kinetic parameters for the enzymic reaction with alpha,alpha-trehalose. The non-selective-inhibition profile for a series of inactive alpha-D-glycopyranosyl phosphates shows that the driving force for the binary-complex formation lies mainly in interactions of the enzyme with the phosphate group and suggests that hydrogen bonding with hydroxyl groups at the catalytic site (subsite -1) contributes to catalysis by providing stabilization, which is specific to the transition state. Vanadate, a tight-binding phosphate mimic, inhibits the phosphorolysis of alpha-D-Glc-1-F by forming a ternary complex whose apparent dissociation constant of 120 microM is approx. 160-fold greater than the dissociation constant of the same inhibitor complex with alpha,alpha-trehalose.