Uridine Diphosphate Glucose Pyrophosphorylase: III. CATALYTIC MECHANISM

Abstract

Abstract Uridine diphosphate glucose pyrophosphorylase, upon incubation with uridine diphosphate glucose or uridine triphosphate, forms a complex which is stable to Sephadex chromatography. Both the uridine portion of uridine triphosphate or uridine diphosphate glucose and the inorganic pyrophosphate or glucose portion of each molecule migrate with the enzyme and cannot be separated by Sephadex chromatography. The amount of substrate bound to the enzyme appears to be dependent upon the stage of aggregation of the protein and has varied experimentally from 5.5 to 10.7 moles per mole of enzyme. Since there are 8 subunits per mole of enzyme, it is likely that 1 mole of substrate is bound per subunit. Uridine monophosphate, inorganic pyrophosphate, and glucose 1-phosphate do not migrate with the enzyme under the same conditions. Glucose 1-phosphate and inorganic pyrophosphate will, however, react with the uridine triphosphate or uridine diphosphate glucose complex and migrate with the enzyme on Sephadex, suggesting the formation of a complex by an exchange reaction. Thus, a compulsory order of substrate binding appears likely. Calf liver pyrophosphorylase has been shown to exist in solution as a complex-polymerizing system of protomers. The environmental factors which control the state of aggregation of the enzyme have not been well defined, but important parameters seem to be specific ions, ionic strength, pH, and concentration of protein. An improved procedure for the purification and crystallization of uridine diphosphate glucose pyrophosphorylase is presented.