PRPP synthetase reaction

Regulation of de novo purine biosynthesis is essential because it consumes a large amount of energy as well as of glycine, glutamine, N °-formyl FH4, and aspartate. Regulation occurs at the PRPP synthetase reaction, the ami- 

Increased levels of intracellular PRPP enhance de novo purine biosynthesis. For example, in patients with HPRT deficiency, the fibroblasts show accelerated rates of purine formation. Several mutations of PRPP synthetase, which exhibit increased catalytic activity with increased production of PRPP, have been described in gouty subjects. 

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Various genetic defects in PRPP synthetase (reaction 1, Figure 34-2) present clinically as gout. Each defect— eg, an elevated increased affinity for ribose 5-... 

PRPP synthetase is an enzyme that catalyzes there reaction below (see here also) ... 

As purines are built on a ribose base (see Fig. 41.2), an activated form of ribose is used to initiate the purine biosynthetic pathway. 5-Phosphoribosyl-l-pyrophosphate (PRPP) is the activated source of the ribose moiety. It is synthesized from ATP and ribose 5 -phosphate (Fig. 41.3), which is produced from glucose through the pentose phosphate pathway (see Chapter 29). The enzyme that catalyzes this reaction, PRPP synthetase, is a regulated enzyme (see section 1I.A.5) however, this step is not the committed step of purine biosynthesis. PRPP has many other uses, which are described as the chapter progresses. 

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. 

PRPP synthetases. A. Enzyme activities in dialyzed hemolysates. Sigmoidal activation is seen for both enzymes. Activity of the mutant enzyme with an increased maximal reaction velocity is, however, increased by a constant proportion at all Pi concentrations. B. Enzyme activities in undialyzed (crude) hemolysates. The mutant enzyme, deficient in inhibitor responsiveness, shows hyperbolic activation with increased activity only at Pi concentrations below 2 mM. C. Enzyme activities in partially purified erythrocyte preparations from a normal individual and the patient with the feedback-resistant PRPP synthetase studied in B. Note hyperbolic activation of the purified normal enzyme and the resulting similarity of the Pi activation curves. D. Enzyme activities in dialyzed fibroblast extracts. The mutant enzyme, with combined increased maximal reaction velocity and diminished nucleotide responsiveness, shows both hyperbolic activation and increased enzyme activity at all Pi concentrations. 

A final category of functional abnormality underlying PRPP synthetase superactivity is represented by the enzyme characterized from the fibroblasts of the child with purine overproduction and deafness referred to earlier. Both increased maximal reaction velocity and... 

We have recently examined the responsiveness to Mg + and MgATP of the subunit aggregates from a superactive form of PRPP synthetase with normal substrate and inhibitor binding properties but an increased maximal reaction velocity. The distribution of forms of the mutant enzyme on sucrose gradient after the dilution and incubation procedures described above was indistinguishable from that of normal PRPP synthetase. In several respects, however, the mutant enzyme behaved in an abnormal fashion. First, under conditions of suboptimal Mg2+... 

Tissues were homogenized in phosphate buffer and sonicated, and the supernatant was used for electrophoresis. Samples were run in phosphate buffer, pH 8.5, on cellulose acetate paper for 2 hours at 4 and at 0.5 mA/cm. PRPP synthetase was located on the paper by a radiochemical assay formation of PRPP from ribose-5-phosphate and ATP was coupled to inosinic acid (IMP) synthesis by the addition to the reaction mixture of labelled hypoxanthine and partially purified hypoxan-thine-guanine phosphoribosyltransferase (HGPRT). 

In this communication we report on the presence of both PRPP synthetase and HGPRT in human platelet lysates. The activity of these enzymes was also studied in rabbit platelets. Platelets were separated and washed according to Ardlie et al. (5) and suspended in tris-buffered saline pH 7.35 (3). The incorporation of purine bases into the nucleotides of intact platelets was investigated by incubating cell suspensions in tris-buffered saline containing labelled purine bases. The reactions were arrested by the addition of perchloric acid and the total nucleotides in the protein-free supernatant were separated from the purine nucleosides and bases by thin... 

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,... 

Fig. 3. The pathway of de novo purine ribonucleotide biosynthesis. The pathway includes the synthesis of PRPP, which is also used in the synthesis of pyrimidines, pyridine nucleotides, histidine, and tryptophan in plants. The enzymes catalyzing the numbered reactions are (1) PRPP synthetase, (2) PRPP amidotransferase, (3) GAR synthetase, (4) GAR transformylase, (5) FGAR amidotransferase, (6) AIR synthetase, (7) AIR carboxylase, (8) succino-AICAR synthetase, (9) adenylosuccinase, (10) AICAR transformylase, and (11) IMP cyclohydrolase.

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]. 

Unlike in purine biosynthesis, the pyrimidine ring is synthesized before it is conjugated to PRPP. The first reaction is the conjugation of carbamoyl phosphate and aspartate to make N-carbamoylaspartate. The carbamoyl phosphate synthetase used in pyrimidine biosynthesis is located in the cytoplasm, in contrast to the carbamoyl phosphate used in urea synthesis, which is made in the mitochondrion. The enzyme that carries out the reaction is aspartate transcarbamoylase, an enzyme that is closely regulated. 

The reaction of carbamoyl phosphate with aspartate [Figure 10.9, reaction (1)] is rate controlling and committing in microorganisms, whereas in mammals carbamoyl phosphate synthetase II seems to the major rate-controlling enzyme. The latter is stimulated by PRPP and purine nucleotides (especially ATP) and... 

The first three reactions are catalyzed by a trifunctional protein which contains carbamoyl-phosphate synthetase II, aspartate carbamoyltransferase and dihydro-orotase. This set of reactions begins with the synthesis of carbamoyl phosphate followed by its condensation with aspartic acid. The third step involves the closure of the ring through the removal of water by the action of dihydro-orotase to yield dihydro-orotate. The fourth enzyme, dihydro-orotate oxidase, oxidizes dihydro-orotate to orotate and is a mitochondrial flavoprotein enzyme located on the outer surface of the inner membrane and utilizes NAD" " as the electron acceptor. The synthesis of UMP from orotate is catalyzed by a bifunctional protein which comprises orotate PRTase and orotidine 5 -phosphate (OMP) decarboxylase. The former phosphoribosylates orotate to give OMP the latter decarboxylates OMP to UMP, the immediate precursor for the other pyrimidine nucleotides. It is interesting to note that whereas five molecules of ATP (including the ATP used in the synthesis of PRPP) are used in the de novo synthesis of IMP, no net ATP is used in the de novo synthesis of UMP. In de novo pyrimidine synthesis, two ATP molecules are used to synthesize carbamoyl phosphate and one ATP is needed to synthesize the PRPP used by orotate PRTase but 3 ATPs... 

The first step in de novo pyrimidine biosynthesis is the synthesis of carbamoyl phosphate from bicarbonate and ammonia in a multistep process, requiring the cleavage of two molecules of ATP. This reaction is catalyzed by carbamoyl phosphate synthetase (CPS), and the bicarbonate is phosphorylated by ATP to form carboxyphosphate and ADP (adenine dinucleotide phosphate). Ammonia then reacts with carboxyphosphate to form carbamic acid. The latter is phosphorylated by another molecule of ATP with the mediation of CPS to form carbamoyl phosphate, which reacts with aspartate by aspartate transcarbamoy-lase to form A-carbamoylaspartate. The latter cyclizes to form dihydroorotate, which is then oxidized by NAD-1- to generate orotate. Reaction of orotate with 5-phosphoribosyl-l-pyrophosphate (PRPP), catalyzed by pyrimidine PT, forms the pyrimidine nucleotide orotidylate. This reaction is driven by the hydrolysis of pyrophosphate. Decarboxylatin of orotidylate, catalyzed by orotidylate decarboxylase, forms uridylate (uridine-5 -monophosphate, UMP), a major pyrimidine nucleotide that is a precursor of RNA (Figure 6.53). 

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