Alpha-retaining glucosyl transfer catalysed by trehalose phosphorylase from Schizophyllum commune: mechanistic evidence obtained from steady-state kinetic studies with substrate analogues and inhibitors.

Fungal trehalose phosphorylase is classified as a family 4 glucosyltransferase that catalyses the reversible phosphorolysis of alpha,alpha-trehalose with net retention of anomeric configuration. Glucosyl transfer to and from phosphate takes place by the partly rate-limiting interconversion of ternary enzyme-substrate complexes formed from binary enzyme-phosphate and enzyme-alpha-d-glucopyranosyl phosphate adducts respectively. To advance a model of the chemical mechanism of trehalose phosphorylase, we performed a steady-state kinetic study with the purified enzyme from the basidiomycete fungus Schizophyllum commune by using alternative substrates, inhibitors and combinations thereof in pairs as specific probes of substrate-binding recognition and transition-state structure. Orthovanadate is a competitive inhibitor against phosphate and alpha-d-glucopyranosyl phosphate, and binds 3 x 10(4)-fold tighter (K(i) approximately 1 microM) than phosphate. Structural alterations of d-glucose at C-2 and O-5 are tolerated by the enzyme at subsite +1. They lead to parallel effects of approximately the same magnitude (slope=1.14; r(2)=0.98) on the reciprocal catalytic efficiency for reverse glucosyl transfer [log (K(m)/k(cat))] and the apparent affinity of orthovanadate determined in the presence of the respective glucosyl acceptor (log K(i)). An adduct of orthovanadate and the nucleophile/leaving group bound at subsite +1 is therefore the true inhibitor and displays partial transition state analogy. Isofagomine binds to subsite -1 in the enzyme-phosphate complex with a dissociation constant of 56 microM and inhibits trehalose phosphorylase at least 20-fold better than 1-deoxynojirimycin. The specificity of the reversible azasugars inhibitors would be explained if a positive charge developed on C-1 rather than O-5 in the proposed glucosyl cation-like transition state of the reaction. The results are discussed in the context of alpha-retaining glucosyltransferase mechanisms that occur with and without a beta-glucosyl enzyme intermediate.