PRPP synthesis

Since biosynthesis of IMP consumes glycine, glutamine, tetrahydrofolate derivatives, aspartate, and ATP, it is advantageous to regulate purine biosynthesis. The major determinant of the rate of de novo purine nucleotide biosynthesis is the concentration of PRPP, whose pool size depends on its rates of synthesis, utilization, and degradation. The rate of PRPP synthesis depends on the availabihty of ribose 5-phosphate and on the activity of PRPP synthase, an enzyme sensitive to feedback inhibition by AMP, ADP, GMP, and GDP. 

The final control mechanism is the inhibition of PRPP synthesis by the allosteric regulation of ribose phosphate pyrophosphokinase. This enzyme is inhibited... 

Phosphoribosyl-l-pyrophosphate (PRPP) synthesis is catalyzed by PRPP synthetase. Note the ribose-5-phosphate for the pathway comes from tlie Pentose Phosphate Pathway (see "PPP/Gluconeogenesis" Lecture). 

The primary site of regulation is Carbamoyl Phosphate Synthetase II (glutamine) which is allosterically inhibited by UTP. Elevated PRPP increases the CPS-II activity to help control PRPP levels. Feedback inhibition (control) is provided by TDP inhibition of PRPP synthesis and UMP inhibition of OMP Decarboxylase. 

The mechanism of the hyperuricemia in most individuals who have gout is unknown. Following is a discussion of biochemical lesions that lead to hyperuricemia and may eventually lead to gout. Enhanced PRPP synthesis results from X-chromosome-linked mutants of PRPP synthetase. Several variants show increased Umax, resistance to feedback inhibition, or a low for ribose 5-phosphate. 

Immune system dysfunction in ADA deficiency has also been ascribed to the inhibition of pyrimidine nucleotide synthesis by adenosine, known as pyrimidine starvation. This may arise from inhibition of conversion of orotic acid to orotidine 5 -monophosphate or from inhibition of PRPP synthesis by excessive synthesis of adenine nucleotides. 

For example, in red cells and ascites cells, PRPP formation cannot depend entirely upon the levels of ribose-5-phosphate because in both types of cells these levels are high enough to saturate PRPP synthetase, yet the amount of PRPP made is not greater than what is observed with much lower levels of ribose-5-phosphate. Tight regulation of PRPP synthesis at the level of synthetase has been demonstrated. 

We shall give only two examples here. First, the fact that the administration of a diet containing 1 % orotic acid to rats depresses the biosynthesis of purine nucleotides possibly by inhibiting PRPP synthesis. Sec-... 

PRPP synthetase superactivity, diversity in the kinetic mechanisms underlying increased PRPP synthesis has been identified. This diversity has important implications for the design of methods for detection of abnormalities of the enzyme. The four categories of kinetic alteration thus far associated with PRPP synthetase superactivity in man are abnormal catalytic properties (increased maximal reaction veloc-city) 2) defective regulatory properties (purine nucleotide feedback resistance) 3) increased affinity for the substrate ribose-5-P and 4) combined alterations of catalytic and regulatory properties. 

Figure 1. Effects of preincubation with Mg2+ and MgATP, singly or in coinbination, on the time course of PRPP synthesis catalyzed by the tetrameric form of PRPP synthetase. Highly purified PRPP synthetase was subjected to sucrose density gradient ultracentrifugation in 1 mM Pi, 1 itiM dithiothreitol (pH 7.4) as previously described. Enzyme sedimenting at 7.1s (corresponding to the 4 subunit aggregated form) was preincubated for 30 minutes at 37 in 1 mM Pi, 1 mM dithiothreitol (pH 7.4) with the additions indicated below. PRPP synthesis was then measured at 26° with final concentrations of 32 mM Pi, 1 mM MgATP,...

Addition of 2,3-DPG to the same system in the amount of 5mM (which is well within the physiological range of this substance in the RBC) resulted in complete obliteration of PRPP synthesis, when tested at 2mM Pi and in the absence of the ATP regenerating system. 

The concentration of PRPP in erythrocytes was measured using the procedure of Sperling et al (5) in which phosphoribosyl-transferase activity present in the haemolysate is utilized. PRPP synthesis during the assay was inhibited by 2,3-diphosphoglyceric acid. Since some of the heterozygotes studied had low HGPRTase activity, PRPP was assayed using the haemolysate adenine phospho-ribosyltransferase activity with radioactive adenine as the second substrate. 

As discussed in the previous section, the synthesis of ribose 5-phosphate must be quite high to provide the ribose 5-phosphate required for de novo purine ribonucleotide biosynthesis. Ribose 5-phosphate required for PRPP synthesis can be synthesized de novo via the oxidative or nonoxidative arms of the pentose phosphate pathway or by recycling of ribose released by the action of nucleotidases/nucleosidases (Fig. 5). The latter pathway requires ribose phosphotransferase (ribokinase), which has been detected in soybean and pea nodule extracts (Christensen and Jochimsen, 1983). The efficient recycling of ribose could eliminate the need for the continuous production of ribose 5-phosphate. Two enzymes of the oxidative branch of the pentose phosphate... 

Fig. 9. Proposed model for the cellular compartmentalization of the reactions of nitrogen fixation, ammonium assimilation, purine synthesis, and ureide biogenesis in infected and uninfected cells of soybean root nodules. Uncertainty still exists with respect to the nature of the intermediate (e.g., IMP, XMP, xanthine, glutamine ) transported from the infected cell to the uninfected cell as well as the site of purine synthesis. In addition, as discussed in the text the site(s) of PRPP synthesis (plastid and/or cytosolic) and the path and site of synthesis (de novo from the PPP or via salvage) of tibose S-phosphate (R-S-P) are s not defined, lliese uncertainties are indicated with question marks and/or dashed lines. Lb, leghemoglobin.

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