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His334 facilitates catalysis by Corynebacterium callunae starch phosphorylase through selective stabilization of the transition state of the reaction, partly derived from a hydrogen bond between its side chain and the C-6 hydroxy group of the glucosyl residue undergoing transfer to and from phosphate. We have substituted His334 by a Gly and measured the disruptive effects of the site-directed replacement on active site function using steady-state kinetics and NMR spectroscopic characterization of the cofactor pyridoxal 5′-phosphate and binding of carbohydrate ligands. Purified H334G showed 0.05% and 1.3% of wild-type catalytic center activity for phosphorolysis of maltopentaose (kcatP = 0.033 s−1) and substrate binding affinity in the ternary complex with enzyme bound to phosphate (Km = 280 mM), respectively. The 31P chemical shift of pyridoxal 5′-phosphate in the wild-type was pH-dependent and not perturbed by binding of arsenate. At pH 7.25, it was not sensitive to the replacement His334→Gly. Analysis of interactions of α-D-glucose 1-phosphate and α-D-xylose 1-phosphate upon binding to wild-type and H334G phosphorylase, derived from saturation transfer difference NMR experiments, suggested that disruption of enzyme–substrate interactions in H334G was strictly local, affecting the protein environment of sugar carbon 6. pH profiles of the phosphorolysis rate for wild-type and H334G were both bell-shaped, with the broad pH range of optimum activity in the wild-type (pH 6.5–7.5) being narrowed and markedly shifted to lower pH values in the mutant (pH 6.5–7.0). External imidazole partly restored the activity lost in the mutant, without, however, participating as an alternative nucleophile in the reaction. It caused displacement of the entire pH profile of H334G by + 0.5 pH units. A possible role for His334 in the formation of the oxocarbenium ion-like transition state is suggested, where the hydrogen bond between its side chain and the 6-hydroxyl polarizes and positions O-6 such that electron density in the reactive center is enhanced.