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

Abstract

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.

Crystallization and X-ray diffraction studies of cellobiose phosphorylase fromCellulomonas uda

“…Their enzymatic versatility is reflected in their classification, as DSPs are found in both glycosyl hydrolase (GH) and glycosyl transferase (GT) families, e.g. chitobiose phosphorylase (GlcNAc-1,4-GlcNAc, inverting, GH94; Honda et al, 2004), maltose phosphorylase (Glc-1,4-Glc, inverting, GH65; Qian et al, 1994) and trehalose phosphorylase (Glc-1,1-Glc, retaining; Nidetzky & Eis, 2001). The ability of DSPs to catalyze reversible reactions arises from the fact that the energy of a glycosylphosphate bond is lower than those of other glycosyl adducts such as glycosyl nucleotides, which are the usual substrates for glycosyl transferases.…”

Studying non-covalent enzyme carbohydrate interactions by STD NMR

“…26 Variations of STD effects between the presence of phosphate and vanadate are small and indicate that both anions cause quite similar binding situations, being in good agreement with previous results. 35 The inhibitory effect of vanadate ions is, therefore, not entirely gained by an influence in binding, which reinforces vanadate to be an excellent model for binding studies at ScTPase. Conclusions about a-glucose (3a) binding pattern in subsite +1 while a-glucose 1-phosphate (7) is also present in the active site cannot be drawn as differences between phosphate and a-glucose 1-phosphate binding are quite pronounced.…”

Crystallization and X-ray diffraction studies of inverting trehalose phosphorylase fromThermoanaerobacter sp.

“…Their enzymatic versatility is reflected in their classification: DSPs are found both in glycosyl hydrolase (GH) and glycosyl transferase (GT) families, e.g. chitobiose phosphorylase (GlcNAc-1,4-GlcNAc, inverting, GH94;Honda et al, 2004), maltose phosphorylase (Glc-1,4-Glc, inverting, GH65; Qian et al, 1994) and trehalose phosphorylase (Glc-1,1-Glc, retaining, GT4; Nidetzky & Eis, 2001). The ability of DSPs to catalyze reversible reactions is a consequence of the fact that the energy of a glycosyl-phosphate bond is lower than in other glycosyl adducts such as glycosyl nucleotides, which are the usual substrate of glycosyl transferases.…”