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

The Biochemical Journal
C Eis, Bernd Nidetzky

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 dur...Continue Reading

References

Sep 15, 1996·European Journal of Biochemistry·N AsanoK Matsui
Oct 6, 1997·FEMS Microbiology Letters·J A JorgeH F Terenzi
Oct 9, 1998·Applied Microbiology and Biotechnology·K SaitoS Horinouchi
Jul 7, 1999·The Biochemical Journal·C Eis, B Nidetzky
Sep 1, 2000·Carbohydrate Research·S J Williams, S G Withers

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