Studies on Cytidine Diphosphate Glucose Pyrophosphorylase and Related Enzymes of Azotobacter vinelandii

Koji Kimata,Sakaru Suzuki

doi:10.1016/s0021-9258(18)96808-0

Abstract

Abstract 1. Formation of 11 sugar nucleotides from the respective nucleoside triphosphates and sugar 1-phosphates have been demonstrated with the crude extract of Azotobacter 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 10-3 m) 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 195-fold. The purified enzyme is specific for both nucleoside triphosphate and sugar 1-phosphate except glucosamine 1-phosphate, which is active in replacing the glucose 1-phosphate. The pH optimum of the reaction is 8.5, and its equilibrium constant measured in the direction of CDP-glucose 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 10-4 m and 7 x 10-4 m, respectively, when the molar ratio of MgCl2 to the substrate is 2:1. 5. The enzyme (Fraction II) is inhibited by its products, CDP-glucose and pyrophosphate. The inhibition by CDP-glucose is strictly competitive with respect to CTP, whereas that by pyrophosphate is noncompetitive. 6. Deoxythymidine triphosphate is a strong inhibitor of the enzyme (Fraction II). It is competitive with CTP only partially, indicating that dTTP combines with a site other than the CTP-binding site. 7. The cytidine nucleotides, isolated from A. vinelandii and tentatively identified as cytidine diphosphate 2-O-methyldeoxyaldose and its carboxylic acid ester, also inhibit the enzyme (Fraction II). Plots of percentage inhibition against inhibitor concentration give sigmoid curves.

Highlights

Introduction1. Formation of 11 sugar nucleotides from the respective nucleoside triphosphates and sugar l-phosphates have been demonstrated with the crude extract of Azofobacfer vinelandii
The CDP-glucose pyrophosphorylase activity has been With the discovery of various sugar nucleotide pyrophosphoresolved into two peaks (Fractions I and II) by diethylamino- rylases, including those that catalyze the transfer of a nucleotidyl ethyl cellulose chromatography
Materiala-CDP-n-glucose, dTDP-n-glucose, GDP-n-glucose, ADP-n-glucose, UDP-n-galactose, and CDP-n-galactose were synthesized from their respective mononucleotide morpholidates and sugar l-phosphates by a slight modification of the method of Roseman et al [7]. n-Ribitol-5-P was prepared by reduction of n-ribose-5-P with NaBH4 [8]. n-Glucosamine-1-P was synthesized by catalytic reduction and deacetylation of l-diphenylphosphoro-3,4,6-tri-o-acetyl-a-n-glucosamine hydrochloride [9]

Summary

Introduction

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. 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 than the CTP-binding site. 7. The cytidine nucleotides, isolated from A. vinelandii and tentatively identified as cytidine diphosphate 2-Omethyldeoxyaldose and its carboxylic acid ester, inhibit the enzyme (Fraction II). Group to a sugar phosphate from the triphosphates of adenosine, 3.

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