Fungal trehalose phosphorylase is classified as a family 4 glucosyltransferase that catalyses the reversible phosphorolysis of α,α-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–α-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 α-d-glucopyranosyl phosphate, and binds 3×104-fold tighter (Ki≈ 1μM) 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; r2 = 0.98) on the reciprocal catalytic efficiency for reverse glucosyl transfer [log (Km/kcat)] and the apparent affinity of orthovanadate determined in the presence of the respective glucosyl acceptor (log Ki). 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 56μM 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 α-retaining glucosyltransferase mechanisms that occur with and without a β-glucosyl enzyme intermediate.
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Research Article|
December 10 2001
α-Retaining glucosyl transfer catalysed by trehalose phosphorylase from Schizophyllum commune: mechanistic evidence obtained from steady-state kinetic studies with substrate analogues and inhibitors
Bernd NIDETZKY;
Bernd NIDETZKY
1
Institute of Food Technology, University of Agricultural Sciences Vienna (BOKU), Muthgasse 18, A-1190 Vienna, Austria
1To whom correspondence should be addressed (e-mail nide@edv2.boku.ac.at).
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Christian EIS
Christian EIS
Institute of Food Technology, University of Agricultural Sciences Vienna (BOKU), Muthgasse 18, A-1190 Vienna, Austria
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Publisher: Portland Press Ltd
Received:
July 10 2001
Revision Received:
September 14 2001
Accepted:
October 19 2001
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2001
2001
Biochem J (2001) 360 (3): 727–736.
Article history
Received:
July 10 2001
Revision Received:
September 14 2001
Accepted:
October 19 2001
Citation
Bernd NIDETZKY, Christian EIS; α-Retaining glucosyl transfer catalysed by trehalose phosphorylase from Schizophyllum commune: mechanistic evidence obtained from steady-state kinetic studies with substrate analogues and inhibitors. Biochem J 15 December 2001; 360 (3): 727–736. doi: https://doi.org/10.1042/bj3600727
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