115] Dephospho-CoA pyrophosphorylase

Abstract

This chapter discusses the determination of dephospho-CoA pyrophosphorylase. This enzyme catalyzes the reversible condensation between ATP and phosphopantetheine to yield dephospho-CoA and inorganic pyrophosphate. The enzyme has not yet been separated from dephospho-CoA kinase; therefore, starting with ATP and phosphopantetheine the product of the over-all reaction is CoA. CoA is measured by means of the phosphotransacetylase assay. Alternatively, the reaction may be measured by the determination of inorganic pyrophosphate. Crude preparations are usually contaminated with ATPase and inorganic pyrophosphatase, however, making such measurements less reliable. In more purified preparations inorganic pyrophosphatase is eliminated, but dephospho-CoA kinase is still present. The latter enzyme displaces the equilibrium in favor of CoA synthesis, and thus the inorganic pyrophosphate level is a direct measure of the condensation reaction. The steps in the purification procedure are: (1) preparation of the crude extract; (2) protamine treatment; (3) ammonium sulfate fractionation; (4) and treatment with calcium phosphate gel. The purified enzyme is quite stable when kept in the frozen state at –10°, no appreciable diminution of activity being noted in three months. Dephospho-CoA pyrophosphorylase is active over the pH range from 6.5 to 8.5 with an optimum near pH 7.5.