Nucleoside diphosphatase (nucleoside diphosphate phosphohydrolase, EC 184.108.40.206) from rat liver has been purified over 800-fold and used for preliminary kinetic studies of the metal ion activation, substrate specificity, and mechanism of action of the enzyme, as well as of its allosteric activation by nucleoside triphosphates. The essential divalent metal ion requirement was met by Mg2+, Mn2+, or Ca2+ and it appears that these ions are involved in the formation of metal-nucleotide complexes that function as substrates for the reaction. At pH 8.5 and a concentration of 0.5 mM, the rate of hydrolysis of the magnesium complexes of various nucleoside diphosphates and thiamine pyrophosphate decreased in the order: uridine diphosphate ≃ guanosine diphosphate ≃ inosine diphosphate ≫ cytosine diphosphate ≥ thiamine pyrophosphate ≫ adenosine diphosphate. No hydrolysis of d-thymidine diphosphate was detected. When MgIDP− was used as the substrate and IDP3− maintained at a concentration of 0.1 mM, double-reciprocal plots of the initial velocity as a function of substrate concentration were curvilinear. Relatively high concentrations of magnesium -nucleoside triphosphates or IDP3− had the effect of increasing the initial reaction velocity at lower substrate concentrations with the result that the double-reciprocal plots became linear. It was concluded that the above results were consistent with the idea that while two molecules of substrate can undergo interdependent reaction with the enzyme, the addition of only one is necessary for reaction to occur. From the data relating to the activating effects of magnesium-nucleoside triphosphates and IDP3−, it has been concluded that these allosteric activators combine with the enzyme at one of the sites at which MgIDP− can react. Higher concentrations of free Mg2+ have been found to inhibit the reaction.
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