Thermal denaturation pathway of starch phosphorylase from Corynebacterium callunae:: Oxyanion binding provides the glue that efficiently stabilizes the dimer structure of the protein

Starch phosphorylase from Corynebacterium callunae is a dimeric protein in which each mol of 90 kDa subunit contains 1 mol pyridoxal 5'-phosphate as an active-site cofactor. To determine the mechanism by which phosphate or sulfate ions bring about a greater than 500-fold stabilization against irreversible inactivation at elevated temperatures (greater than or equal to 50 degrees C), enzyme/oxyanion interactions and their role during thermal denaturation of phosphorylase have been studied. By binding to a protein site distinguishable from the catalytic site with dissociation constants of K-sulfate = 4.5 mM and K-phosphate approximate to 16 mM, dianionic oxyanions induce formation of a more compact structure of phosphorylase, manifested by (a) an increase by about 5% in the relative composition of the alpha-helical secondary structure, (b) reduced H-1/H-2 exchange, and (c) protection of a cofactor fluorescence against quenching by iodide. irreversible loss of enzyme activity is triggered by the release into solution of pyridoxal 5'-phosphate, and results from subsequent intermolecular aggregation driven by hydrophobic interactions between phosphorylase subunits that display a temperature-dependent degree of melting of secondary structure. By specifically increasing the stability of the dimer structure of phosphorylase (probably due to tightened intersubunit contacts), phosphate, and sulfate, this indirectly (1) preserves a functional active site up to approximate to 50 degrees C, and (2) stabilizes the covalent protein cofactor linkage up to approximate to 70 degrees C. The effect on thermostability shows a sigmoidal and saturatable dependence on the concentration of phosphate, with an apparent binding constant at 50 degrees C of approximate to 25 mM. The extra stability conferred by oxyanion-ligand binding to starch phosphorylase is expressed as a dramatic shift of the entire denaturation pathway to a approximate to 20 degrees C higher value on the temperature scale.