Phosphoribosylpyrophosphate synthetase

The synthesis of phosphoribosylpyrophosphate from ATP and ribose-5-phosphate is catalyzed by phosphoribosylpyrophosphate synthetase. Superactivity of this enzyme may be a cause of overproduction of uric acid, which may result in gout. 

Assay of the enzyme is based on the separation of ATP, ADP, and AMP on a Tosoh ODS-120A column (4.6 mm x 250 mm, 5 fitn). The mobile phase 

The reaction mixture contained 40 mAf sodium phosphate buffer (pH 7.4), 1 mM ribose-5-phosphate, 1.4 mM ATP, 6 mM MgCl2,1 mA/ reduced glutathione, and 1.8 IU/mL adenylate kinase in a final volume of 2.0 mL. The reaction was started by adding ATP. After further incubation at 37°C, 0.5 mL of the reaction mixtures was withdrawn at 10 and 30 minutes and added to tubes containing 0.5 mL of 0.6 M perchloric acid. After centrifugation, the supemate was neutralized by adding an equal volume of 0.4 M Na2HP04, and aliquots were used for HPLC analysis. Product ADP increased linearly with time for 40 minutes. 

The enzyme source was thrice-washed red blood cells that were then lysed by freeze-thawing, treated with charcoal and dextran, and centrifuged. The supernates were used as enzyme samples. 

SURVEY OF ENZYMATIC ACTIVITIES ASSAYED BY THE HPLC METHOD 

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Glucose was first converted enzymatically into ribose 5 -phosphate from which GMP was subsequently obtained by the action of phosphoribosylpyrophosphate synthetase and guanosine phos-phoribosyl transferase. A two-step phosphorylation to GTP followed by treatment with recombinant GTP-cyclohydrolase I from E. coli gave (51) <90Ml 718-12). This method also allows l4C-labeling in the 6- and 7-position as well as the carbon sidechain. 

The first ATP-dependent synthetase to be subjected to analysis for substitution stereochemistry was phosphoribosylpyrophosphate synthetase [78]. This analysis was novel in that it utilized a coordination exchange-inert Co-ATP complex for this purpose and circular dichroic analysis for relative configurations of substrate and product. The reaction of Equation 16 was catalyzed by this enzyme. 

Figure 9.118 HPLC profiles of the reaction mixtures after the enzyme reaction of erythrocyte phosphoribosylpyrophosphate synthetase from a healthy subject. Incubation times were 10 minutes (a) and 30 minutes (fc). (From Sakuura, et al., 1991.)...

G5. Green, C. D., and Martin, D. W., Jr., Characterization of a feedback-resistant phosphoribosylpyrophosphate synthetase from cultured, mutagenized hepatoma cells that overproduce purines. Proc. Nat. Acad. Set. V.S. 70, 3698--3702 (1973). 

Sperling, O., Boer, P., Persky-Brosh, S., Kanarek, E., and De Vries, A., Altered kinetic property of erythroc e phosphoribosylpyrophosphate synthetase in excessive purine production, flee. Eur. Etud. Clin. Biol 17, 703-706 (1972). 

Wyngaarden, J.B. and Kelley, W.N. Gout with purine overproduction due to increased phosphoribosylpyrophosphate synthetase activity. In Gout and Hyperuricemia, Chapter 24, Grune Stratton, New York, 1976, pp. 301-308. 

PHOSPHORIBOSYLPYROPHOSPHATE SYNTHETASE SUPERACTIVITY DETECTION, CHARACTERIZATION OF UNDERLYING DEFECTS, AND TREATMENT... 

M. A. Becker, K. O. Raivio, B. Bakay, W. B. Adams and W. L. Nyhan. Variant human phosphoribosylpyrophosphate synthetase altered in regulatory and catalytic functions. J.Clin.Invest. 65 109 (1980). 

The patient s RBC ADA activity was 43 000 nmol.min . ml RBC " (normal values 495 i 60), There was an about 3-fold increase of red cell pyrimidine 5 -nucleotidase and orotate phosphoribosyl-transferase, whereas other enzymes of purine and pyrimidine metabolism (inosine phosphorylase, adenosine kinase, adenine phosphoribo-syltransferase, hypoxanthine-guanine-phosphoribosyltransferase, phosphoribosylpyrophosphate synthetase) were normal or slightly elevated. There was a 6-fold increase of pyruvate kinase activity relatively to comparably reticulocyte-rich blood, and a 1.5 to 3-fold increase of the other enzymatic activities of glucose and glutathione metabolism. Plasma ADA was much elevated (30.5 pmol.min . ml normal value 5.1 - 2.5), probably reflecting intravascular hemolysis. ADA activity in lymphocytes (2.13 nmol.min 1.10 cells normal 1.93 0.61) and in fibroblasts (26 nmol.min l.mg protein 1 normal range 14-118) was normal, whereas the small increase of activity in platelets (59.5 nmol.min . 10 cells control 26.7) and in the liver (8.4 pmol.min . mg protein" normal ... 

Sperling, 0., P. Boer, S. Persky-Brosh, E. Kanarek, and A. de Vries. 1972. Altered kinetic property of erythrocyte phosphoribosylpyrophosphate synthetase in excessive purine production. Europ. J. Clin. Biol. Res. 17 703. 

Becker, M. A., L. J. Meyer, A. W. Wood, and J. E. Seegmiller. 1973. Purine overproduction in man associated with increased phosphoribosylpyrophosphate synthetase activity. Science 179 1123. 

Adenine- and hypoxanthine-guanine phosphoribosyl-transferase in hemolysates were within the normal range Phosphoribosylpyrophosphate synthetase activity in the hemolysate of the hyperuricemic patient was increased ... 

ELECTROPHORETIC SEPARATION OF NORMAL AND MUTANT PHOSPHORIBOSYLPYROPHOSPHATE SYNTHETASE... 

Table 23.8. Phosphoribosylpyrophosphate synthetase superactivity [Patients 24 (world)]...

Phosphoglycerate dehydrogenase deficiency Phosphomannomutase 2 deficiency Phosphomannose isomerase deficiency Phosphoribosylpyrophosphate synthetase deficiency Phytanyl-CoA hydroxylase deficiency PKU... 

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