Fungal trehalose phosphorylase: kinetic mechanism, pH-dependence of the reaction and some structural properties of the enzyme from Schizophyllum commune

Abstract

Initial-velocity measurements for the phospholysis and synthesis of α,α-trehalose catalysed by trehalose phosphorylase from Schizophyllum commune and product and dead-end inhibitor studies show that this enzyme has an ordered Bi Bi kinetic mechanism, in which phosphate binds before α,α-trehalose, and α--glucose is released before α--glucose 1-phosphate. The freeenergy profile for the enzymic reaction at physiological reactant concentrations displays its largest barriers for steps involved in reverse glucosyl transfer to -glucose, and reveals the direction of phospholysis to be favoured thermodynamically. The pH dependence of kinetic parameters for all substrates and the dissociation constant of -glucal, a competitive dead-end inhibitor against -glucose (K i l 0.3 mM at pH 6.6 and 30 mC), were determined. Maximum velocities and catalytic efficiencies for the forward and reverse reactions decrease at high and low pH, giving apparent pK values of 7.2-7.8 and 5.5-6.0 for two groups whose correct protonation state is required for catalysis. The pH dependences of k cat \K are interpreted in terms of

Inductively coupled plasma- and glow discharge plasma-sector field mass spectrometry : Part I. Tutorial: Fundamentals and instrumentation

“…This is due to the very high neutron cross-section of 10 B, which amounts 3.84 Â 10 3 barns and therefore exceeds 8 Â 10 5 times that of 11 B. 122 In science, enriched isotopes are used for tracer experiments in biology and medicine, [123][124][125][126] or quantification in analytical chemistry applying isotope dilution mass spectrometry. 127,128 Since effects 2 and 3 affect all isotopes of an element and lead to a shift in the isotopic composition, this process is denoted as isotope fractionation.…”

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

“…[6,7,11] and has been shown by peptide mass mapping to share close structural similarity with the classified trehalose phosphorylase sequences [7]. It utilizes a steady-state ordered Bi Bi kinetic mechanism as shown in Scheme 1.…”

“…It has therefore attracted much interest in regulation and mechanism of action of α,α-trehalose-converting enzymes in general and trehalase in particular [1,[3][4][5]. Recent studies have revealed clearly the involvement of another enzyme trehalose phosphorylase in catabolism of α,α-trehalose in fungi [2,6,7]. Trehalose phosphorylases (EC 2.4.1.64) catalyse degradation of α,α-trehalose with net retention of anomeric configuration and by using phosphate as the ultimate glucosyl acceptor [8].…”

“…The active site of trehalose phosphorylase contains two separate binding sites for the substrates phosphate and α,α-trehalose. The α,α-trehalosebinding site is composed of two subsites, each of which interacts with one constituent glucosyl residue of the disaccharide substrate [7,11]. Ternary-complex conversion and release of Glc-1-P are major rate-limiting steps for the phosphorolysis direction of the reaction [7].…”

“…Hence, α--glucose-1-phosphate (Glc-1-P) and -glucose are the reaction products. Trehalose phosphorylases are glucosyl transferases which, in spite of the presence of 55 M water, synthesize glycosidic bonds without the occurrence of detectable error hydrolysis [6,7]. Based on similarity at the level of primary structure, trehalose phosphorylases from Grifola frondosa (GenBank2 accession no.…”