Oxyanion-mediated protein stabilization: Differential roles of phosphate for preventing inactivation of bacterial alpha-glucan phosphorylases

Maltodextrin phosphorylase (MalP) from Escherichia coli and starch phosphor ylase (StP) from Corynebacterium callunae are significantly stabilized in t he presence of phosphate against inactivation by elevated temperature or ur ea. The stabilizing effect of phosphate was observed at ion concentrations below 50 mM. Therefore, it is probably due to preferential binding of phosp hate to the folded conformations of the phosphorylases. For StP, phosphate binding inhibited the dissociation of the active-site cofactor pyridoxal 5' -phosphate. Phosphate-liganded StP was at least 500-fold more stable at 60 degreesC than the free enzyme at the same temperature. It showed an apparen t transition midpoint of 5.2 M for irreversible denaturation by urea, and t his midpoint was increased by a denaturant concentration of 4 M relative to the corresponding transition midpoint of free StP in urea. The mechanisms of inactivation and denaturation of MalP at 45 degreesC and by urea involve formation of a cofactor-containing, insoluble protein aggregate. Under den aturing conditions, phosphate was shown to inhibit aggregation of the rever sibly inactivated MalP dimer.