Inhibition of PRPP synthetase

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. 

Inhibition by AMP and GMP, is competitive with respect to PRPP. The human placental enzyme exists in a small form (M.W. 133,000) and a large form (M.W. 270,000). The small form is catalytically active. Ribonucleotides convert the active form to the large form, whereas PRPP does the opposite. The regulatory actions of PRPP synthetase and amidophosphoribosyltransferase are coordinated. When there is a decrease in the intracellular concentration of adenine ribonucleotides, PRPP synthetase is activated this results in increased synthesis of PRPP, which in turn converts the inactive form of amidophosphoribosyltransferase to the active form and increases production of purine nucleotides. 

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. 

An increased production of uric acid can result from clinical conditions in which there is a rapid increase in the rate of degradation of purine nucleotides. This degradation occurs as a result of the turnover or breakdown of nucleic acids and soluble nucleotides in the cell often associated with breakdown of the cell itself. Examples of this would include the acute leukemias and hemolytic anemias (2). In addition, the degradation of purine nucleotides can occur as a result of alterations in the energy of the cell which enhance the breakdown of ATP. Examples of this might include starvation, muscular exertion, and hypoxia. In some of these latter conditions related to the catabolism of purine nucleoside triphosphates, there may also be compensatory increase in the rate or purine biosynthesis de novo related to the release of feedback inhibition at the level of PRPP synthetase and/or PRPP amidotransferase. 

Essentially similar results were obtained, when glucose was replaced by Rib-5-P. The parallel behavior of the two substrates seemed, therefore, to support the notion that the site of inhibition was located at the stage of PRPP synthetase. It may be pertinent to refer in this context to a closely analogous control mechanism operating at the level of hexokinase which has been described by Brewer. ... 

The first step of this sequence, which is not unique to de novo purine nucleotide biosynthesis, is the synthesis of 5-phosphoribosylpyrophosphate (PRPP) from ribose-5-phosphate and adenosine triphosphate. Phosphoribosyl-pyrophosphate synthetase, the enzyme that catalyses this reaction [278], is under feedback control by adenosine triphosphate [279]. Cordycepin interferes with thede novo pathway [229, 280, 281), and cordycepin triphosphate inhibits the synthesis of PRPP in extracts from Ehrlich ascites tumour cells [282]. Formycin [283], probably as the triphosphate, 9-0-D-xylofuranosyladenine [157] triphosphate, and decoyinine (LXXlll) [284-286] (p. 89) also inhibit the synthesis of PRPP in tumour cells, and this is held to be the blockade most important to their cytotoxic action. It has been suggested but not established that tubercidin (triphosphate) may also be an inhibitor of this reaction [193]. 

PRPP is an "activated pentose" that participates in the synthesis of purines and pyrimidines, and in the salvage of purine bases (see p. 294). Synthesis of PRPP from ATP and ribose 5-phosphate is catalyzed by PRPP synthetase (ribose phosphate pyrophosphokinase, Figure 22.6). This enzyme is activated by inorganic phosphate (Pi) and inhibited by purine nucleotides (end-product inhibition). [Note The sugar moiety of PRPP is ribose, and therefore ribonucleotides are the end products of de novo purine synthesis. When deoxy-ribonucleotides are required for DNA synthesis, the ribose sugar moiety is reduced (see p. 295).]... 

The answer is c. (Ivlurray, pp 375— /O I. Scrivt i, pp 2513—2570. Sack, pp 121—138. Wilson, pp 287—320.1 Several control sites exist in the path of purine synthesis where feedback inhibition occurs, AMP, GMP, or IMP may inhibit the first step of the pathway, which is the synthesis ol 5-phosphoribosyl-l-pyrophosphate (PRPP). PRPP synthetase is specifically inhibited. All three nucleotides can inhibit glutamine PRPP aminotranslerase, which catalyzes the second step of the. pathway. AMP blocks the conversion ol IMP to adenylosuccinate. GMP inhibits the lormation ol xanthylate Irom IMP Thus, blockage rather than enhancement ol IMP metabolism to AMP and GMP effectively inhibits purine biosynthesis. 

PRPP is an important intermediate in the de novo synthesis of purines pathway (Figure 22.4). Defects in PRPP synthetase may render it insensitive to feedback inhibition by purine nucleotides. Thus, purine nucleotides are overproduced, leading to excessive uric acid synthesis and gout (Figure 22.9). 

A primary site of regulation is the synthesis of PRPP. PRPP synthetase is negatively affected by GDP and, at a distinct allosteric site, by ADR Thus, the simultaneous binding of an oxypurine (eg., GDP) and an aminopurine (eg., ADP) can occur with the result being a synergistic inhibition of the enzyme. This enzyme is not the committed step of purine biosynthesis PRPP is also used in pyrimidine synthesis and both the purine and pyrimidine salvage pathways. 

Fig. 41.9. The regulation of purine synthesis. PRPP synthetase has two distinct allosteric sites, one for ADP, the other for GDP. Glutamine phosphoribosyl amidotransferase contains adenine nucleotide and guanine nucleotide binding sites the monophosphates are the most important, although the di- and tri-phosphates will also bind to and inhibit the enzyme. Adenylosuccinate synthetase is inhibited by AMP IMP dehydrogenase is inhibited by GMP.

A number of compounds are capable of altering the rate of purine biosynthesis Je novo. Many that are capable of inhibiting purine biosynthesis Je novo appear to do so as a result of depletion of intracellular levels of PRPP (7). In most of these cases, the nucleotide derivative of the compound is formed and this derivative may also play a role in the inhibition of the PRPP amidotransferase and/or PRPP synthetase. Examples include adenine, allopurinol, 2 6-diaminopurine, nicotinic acid, and orotic acid. On the other hand, several compounds have been studied which lead to an acceleration in the rate of purine biosynthesis apparently mediated by an increased level of PRPP (7). Examples of this category might include fructose, methylene blue, ACTH, TSH, and estrogens. 

Effect of growth in adenine on PRPP synthetase and amidophosphoribosyltransferase If adenine inhibited de novo purine biosynthesis by a repression type mechanism, the specific... 

Effect of actinomyin JD, cycloheximide, and azacytidine on de novo purine biosynthesis Table 2 illustrates that all three inhibitors, actinomycin D, cycloheximide, and azacytidine inhibit de novo purine biosynthesis as measured by the incorporation of -formate into total purines Labelling was for 2 hours, and the results are the average of three experiments Controls had between 25,000 and 30,000 cpm/10 cells Actinomycin D had no effect on Li vitro assayed PRPP amidotransferase or PRPP synthetase activities for cell pretreated for 2 hours with the drug ... 

Phosphoribosylpyrophosphate (PRPP) synthetase (E.C. 2.7.6.1) catalyzes the formation of PRPP from ribose-5-phosphate and ATP in the presence of Mg and inorganic phosphate. The product, PRPP, is a substrate of the first rate limiting step of the de novo synthesis of purine nucleotides and its availability has been shown to regulate this pathway in human tissue (l). A superactive mutant erythrocyte PRPP synthetase with decreased sensitivity to feedback inhibition has recently been found by us in a gouty family (2,3). 

DPG. In order to elucidate whether the hyperbolic response of the mutant PRPP synthetase to increasing phosphate concentration in hemolysate reflects an abnormal response to inhibitors, a system devoid of inhibitors was employed. Using stroma-free charcoal-adsorbed hemolysate treated with DEAE-cellulose, the difference in reaction to increasing inorganic phosphate concentration between the mutant enzyme and the normal enzyme disappeared both exhibiting a hyperbolic response (Fig. 2). It was furthermore found that the mutant enzyme had a decreased sensitivity to inhibition by GDP, ADP,... 

Pig. 3. Inhibition of partially purified normal and mutant PRPP synthetase enzymes by GDP, ADP, 2—3DPG and AMP. Concentration of inorganic phosphate was 1 mM. o control enzyme mutant enzyme. 

It thus appears that the mutant PRPP synthetase enzyme is structurally altered in such a way that only its regulatory properties but not its catalytic properties are affected. This selective alteration proves that these two properties are located at different sites, the enzyme being allosteric. Examples are known of mutations in bacteria (18,19) and Ehrlich ascites cells (20) which altered the susceptibility of regulatory enzymes to effector mulecules. An indication for such a mutation in man has been obtained by Henderson et al in studies on fibroblasts from two patients with purine overproduction and gout, showing reduced effectiv-ness of product inhibition of purine biosynthesis (2l). 

The committed step of this pathway is the synthesis of carbamoyl phosphate from glutamine and CO2, catalyzed by carbamoyl phosphate synthetase II. This enzyme is inhibited by UTP and activated by ATP and PRPP. 

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