Catalytic mechanism of α-retaining glucosyl transfer by Corynebacterium callunae starch phosphorylase: the role of histidine-334 examined through kinetic characterization of site-directed mutants

Abstract

Purified site-directed mutants of Corynebacterium callunae starch phosphorylase in which His-334 was replaced by an alanine, glutamine or asparagine residue were characterized by steady-state kinetic analysis of enzymic glycosyl transfer to and from phosphate and studies of ligand binding to the active site. Compared with wild-type, the catalytic efficiencies for phosphorolysis of starch at 30 °C and pH 7.0 decreased approx. 150- and 50-fold in H334Q (His334→Gln) and H334N mutants, and that of H334A was unchanged. In the direction of α-glucan synthesis, selectivity for the reaction with G1P (α-D-glucose 1-phosphate) compared with the selectivity for reaction with α-D-xylose 1-phosphate decreased from a wild-type value of ∼20000 to 2600 and 100 in H334N and H334Q respectively. Binding of G1P to the free enzyme was weakened between 10-fold (H334N, H334Q) and 50-fold (H334A) in the mutants, whereas binding to the complex of enzyme and α-glucan was not affected. Quenching of fluorescence of the pyridoxal 5′-phosphate cofactor was used to examine interactions of the inhibitor GL (D-gluconic acid 1,5-lactone) with wild-type and mutant enzymes in transient and steady-state experiments. GL binding to the free enzyme and the enzyme–phosphate complex occurred in a single step. The 50-fold higher constant (Kd) for GL dissociation from H334Q bound to phosphate resulted from an increased off-rate for the ligand in the mutant, compared with wild-type. A log-log correlation of catalytic-centre activity for phosphorolysis of starch with a reciprocal Kd value established a linear free-energy relationship (slope=1.19±0.07; r2=0.991) across the series of wild-type and mutant enzymes. It reveals that GL in combination with phosphate has properties of a transition state analogue and that the His-334 side chain has a role in selectively stabilizing the transition state of the reaction.