Purine PRPP synthetase

Primary gout can be caused by overproduction of purine catabolites due to X-linked mutations of PRPP synthetases that render the enzyme insensitive to allosteric inhibitors. 

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).]... 

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-... 

PRPP synthetase requires inorganic phosphate as an allosteric activator. Its activity depends on intracellular concentrations of several end products of pathways in which PRPP is substrate. These end products are purine and pyrimidine nucleotides (Figure 27-12). 

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. 

Feedback regulation of the de novo pathway of purine biosynthesis. Solid lines represent metabolic pathways, and broken lines represent sites of feedback regulation. , Stimulatory effect , inhibitory effect. Regulatory enzymes A, PRPP synthetase B, amidophosphoribosyltransferase C, adenylosuccinate synthetase D, IMP dehydrogenase. 

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 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. 

Two enzyme abnormalities resulting in an overproduction of uric acid have been well described (Fig. 91-1). The first is an increase in the activity of phosphoribosyl pyrophosphate (PRPP) synthetase, which leads to an increased concentration of PRPP. PRPP is a key determinant of purine synthesis and thus uric acid production. The second is a deficiency of hypoxanthine guanine phosphoribosyl transferase (HGPRT). 

PURINE NUCLEOTIDES The de novo synthesis of purine nucleotides begins with the formation of 5-phospho-a-D-ribosyl- 1-pyrophosphate (PRPP) catalyzed by ribose-5-phosphate pyrophosphokinase (PRPP synthetase). 

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). 

The regulation of purine nucleotide biosynthesis occurs at four points in the pathway. The enzymes PRPP synthetase, amidophosphoribosyl transferase, IMP... 

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. 

Regulation of purine synthesis occurs at several sites (Fig. 41.9). Four key enzymes are regulated PRPP synthetase, amidophosphoribosyl transferase. 

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.

Previous attempts to address this problem have shown no increase in specific activity of PRPP synthetase or amidophosphoribosyitransferase under conditions of stimulation of purine production (6). But in these studies enzyme assays were conducted under activating conditions in the assay cuvette. The present study (10) demonstrates that marked changes of enzyme activity can be brought about in vivo by allosteric effects that would not be detected by routine assays and that require conditions which allow recognition of the physical and catalytic state of the enzyme in the tissue at the moment of analysis. 

Fig, 1 Outline of purine metabolism. (1) PRPP amidotransferase (2) hypox Anithine-guanine ribosyltransferase (3) PRPP synthetase (4) adenine phosphoribosyltransferse (5) adenosine de nase (6) purine nucleoside phosphorylase (7) 5 -nucleotidase (8) xanthine oxidase. 

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. 

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. 

No evidence for the presence of a known enzyme abnormality causing purine overproduction could be obtained. The erythrocyte activity of hypoxanthine-guanine phos-phoribosyltransferase (HGPRT), of adenine phosphoribosyltransferase (APRT), and of phosphoribosylpyrophosphate (PRPP) synthetase were all in the normal range. Erythrocyte PRPP generation, as well as the acitivity of the pentose phosphate pathway was also normal (Table 1). In addition, the rate of de novo synthesis of purine nucleotides in cultured skin fibroblasts from the patient was found to be normi. 

Since the initial report by Drs. Sperling and de Vries and their colleagues, inherited superactivity of PRPP synthetase has become established as an unusual cause of purine overproduction, hyperuricemia, and gout in man. To date, detailed investigations of 7 families with superactive PRPP synthetase have been published, and I am aware of 4 additional families currently under study. In each family, the index cases have been males, and where studied, the patterns of inheritance of the enzyme aberrations have been consistent with X-linked transmission, reflecting the apparent structural basis of superactivity in each defective enzyme. 

Evidence of purine overproduction in childhood has led to detection of superactive PRPP synthetases in 2 families which are of special interest for several reasons, First, the hemizygous affected males in these families show severe sensorineural deafness in addition to uric acid overproduction. Second, the mothers of these boys share both the metabolic and hearing abnormalities with their sons, and one of these women has had both acute gouty arthritis and uric acid urolithiasis. Finally, as discussed below, the functional derangement in the enzyme of one of the families is unusually marked with more severe metabolic consequences of PRPP synthetase superactivity which might explain the childhood clinical onset, the development of gout in the mother, and even the associated deafness. 

Despite structural diversity in the superactive enzymes of individual families, studies of PRPP and purine metabolism carried out both vivo and in cells cultured from affected hemizygous males support the idea that a common mechanism accounts for the association of PRPP synthetase superactivity with uric acid overproduction. Increased intracellular PRPP concentrations and rates of PRPP generation as well as increased rates of all PRPP-dependent purine nucleotide synthetic processes are constant accompaniments of enzyme superactivity. These findings suggest a scheme to explain the association of the enzyme defect with uric acid overproduction PRPP synthetase superactivity -> increased intracellular PRPP generation and concentration > increased rate of purine nucleotide synthesis excessive uric acid synthesis. 

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. 

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... 

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