Studies on cytidine diphosphate glucose pyrophosphorylase and related enzymes of Azotobacter vinelandii.

1. Formation of 11 sugar nucleotides from the respective nucleoside triphosphates and sugar l-phosphates have been demonstrated with the crude extract of Azofobacfer vinelandii. These are adenosine diphosphate glucose, cytidine diphosphate glucose, uridine diphosphate glucose, deoxythymidine diphosphate glucose, adenosine diphosphate N-acetylglucosamine, uridine diphosphate N-acetylglucosamine, deoxythymidine diphosphate N-acetylglucosamine, adenosine diphosphate glucosamine, cytidine diphosphate glucosamine, uridine diphosphate glucosamine, and deoxythymidine diphosphate glucosamine. 2. The CDP-glucose pyrophosphorylase activity has been resolved into two peaks (Fractions I and II) by diethylaminoethyl cellulose chromatography. 3. One of these fractions (Fraction I: Michaelis constant (Km) for cytidine triphosphate = 3.3 X 10e3 H) is accompanied by a predominant UDP-glucosamine pyrophosphorylase activity (Km for uridine triphosphate = 1.9 X 10-4 M), and the ratio of the two activities (1:26) did not vary during the purification and inactivation at 50’. It seems likely that both activities are catalyzed by the same enzyme. 4. Another fraction (Fraction II) has been purified 195fold. The purified enzyme is specific for both nucleoside triphosphate and sugar l-phosphate except glucosamine l-phosphate, which is active in replacing the glucose l-phosphate. The pH optimum of the reaction is 8.5, and its equilibrium constant measured in the direction of CDPglucose synthesis is 0.57. The enzyme absolutely requires Mg++ ions for activity. Variation of the Mg* ion concentration relative to either pyrophosphate or CTP has a marked influence on the reaction velocity as well as Km values for the substrates. Km values calculated for pyrophosphate and CTP are 5.6 X 10e4 M and 7 X 10v4 M, respectively, when the molar ratio of MgClz to the substrate is 2:l. 5. The enzyme (Fraction II) is inhibited by its,products, CDP-glucose and pyrophosphate. The inhibition by CDPglucose is strictly competitive with respect to CTP, whereas that by pyrophosphate is noncompetitive. 6. Deoxythymidine triphosphate is a strong inhibitor of the enzyme (Fraction H). It is competitive with CTP only