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 a n active-site cofactor. To determine the mechanism by which phosphate or su lfate ions bring about a greater than 500-fold stabilization against irreve rsible inactivation at elevated temperatures (greater than or equal to 50 d egrees C), enzyme/oxyanion interactions and their role during thermal denat uration of phosphorylase have been studied. By binding to a protein site di stinguishable from the catalytic site with dissociation constants of K-sulf ate = 4.5 mM and K-phosphate approximate to 16 mM, dianionic oxyanions indu ce 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 enz yme activity is triggered by the release into solution of pyridoxal 5'-phos phate, and results from subsequent intermolecular aggregation driven by hyd rophobic interactions between phosphorylase subunits that display a tempera ture-dependent degree of melting of secondary structure. By specifically in creasing the stability of the dimer structure of phosphorylase (probably du e to tightened intersubunit contacts), phosphate, and sulfate, this indirec tly (1) preserves a functional active site up to approximate to 50 degrees C, and (2) stabilizes the covalent protein cofactor linkage up to approxima te to 70 degrees C. The effect on thermostability shows a sigmoidal and sat uratable dependence on the concentration of phosphate, with an apparent bin ding constant at 50 degrees C of approximate to 25 mM. The extra stability conferred by oxyanion-ligand binding to starch phosphorylase is expressed a s a dramatic shift of the entire denaturation pathway to a approximate to 2 0 degrees C higher value on the temperature scale.