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-d-glucose is released before α-d-glucose 1-phosphate. The free-energy profile for the enzymic reaction at physiological reactant concentrations displays its largest barriers for steps involved in reverse glucosyl transfer to d-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 d-glucal, a competitive dead-end inhibitor against d-glucose (Ki = 0.3mM at pH6.6 and 30°C), 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 kcat/K are interpreted in terms of monoanionic phosphate and α-d-glucose 1-phosphate being the substrates, and of the pK value seen at high pH corresponding to the phosphate group in solution or bound to the enzyme. The Ki value for the inhibitor decreases outside the optimum pH range for catalysis, indicating that binding of d-glucal is tighter with incorrectly ionized forms of the complex between the enzyme and α-d-glucose 1-phosphate. Each molecule of trehalose phosphorylase contains one Mg2+ that is non-dissociable in the presence of metal chelators. Measurements of the 26Mg2+/24Mg2+ ratio in the solvent and on the enzyme by using inductively coupled plasma MS show that exchange of metal ion between protein and solution does not occur at measurable rates. Tryptic peptide mass mapping reveals close structural similarity between trehalose phosphorylases from basidiomycete fungi.

Puzzling over orphan enzymes

“…In the fungus Schizophyllum commune, tryptic peptide mass mapping disclosed a significant portion of the sequence of the trehalose phosphorylase (EC 2.4.1.231), opening the way to find close structural similarities with homologues in other basidiomycete fungi [29]. The availability of genomic sequences is crucial to going a step further.…”

Increased trehalose biosynthesis improves Mesorhizobium ciceri growth and symbiosis establishment in saline conditions

“…This pathway has been reported in some fungi and protists (Eis et al 2001;Han et al 2003;Paul et al 2008). More recently, a new alternative pathway for trehalose synthesis and degradation via trehalose glycosyltransferring synthase (encoded by gene treT) was reported.…”

The phosphate site of trehalose phosphorylase from Schizophyllum commune probed by site-directed mutagenesis and chemical rescue studies

“…These pH effects are explicable on account of changes in the ionization states of the enzyme and the substrate phosphate (pK a,2 = 7.2) and, in the case of K512A, deprotonation of propargylamine at high pH (pK a = 8.2). Analysis of pH-rate profiles for wild-type ScTPase suggested that H 2 PO 4 ) is the protonic form of phosphate utilized in the enzymatic reaction [17]. The pH-dependence of functional complementation of K512A was therefore examined in more detail.…”

“…In the direction of phosphorolysis, recruitment of phosphate by the free enzyme induces binding recognition for a,a-trehalose and promotes the catalytic steps of glucosyl transfer. d-glucose is released from the ternary enzyme-product complex, and dissociation of G1P regenerates the free enzyme [17]. The unreactive phosphate-analogue vanadate is a transition state-like inhibitor of ScTPase [18,19] whereby partial mimicry of the transition state was proposed to derive from a hydrogen bond between vanadate and the a-anomeric hydroxyl of the glucose leaving group ⁄ nucleophile (Fig.…”

“…Schizophyllum commune trehalose phosphorylase (ScTPase; EC 2.4.1.231) utilizes a glycosyltransferaselike catalytic mechanism to convert a,a-trehalose and phosphate into a-d-glucose 1-phosphate (G1P) and a-d-glucose in a freely reversible reaction [16,17]. In the direction of phosphorolysis, recruitment of phosphate by the free enzyme induces binding recognition for a,a-trehalose and promotes the catalytic steps of glucosyl transfer.…”