James B. Wyngaarden

Regulation of purine biosynthesis in cultured human cells. I. Effects of orotic acid.

Abstract Orotic acid, an intermediate in the synthesis of pyrimidine nucleotides, has been shown in the present study to inhibit an early step of purine biosynthesis de novo in cultured human cells as manifested by the decreased synthesis of N-formylglycinamide ribonucleotide (FGAR). Concentrations of orotic acid that inhibited purine synthesis also reduced intracellular levels of 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P), an essential substrate for the first enzyme unique to this pathway. Orotic acid had no inhibitory effect on FGAR synthesis in mutant cells with high PP-ribose-P levels or in normal cells if depletion of PP-ribose-P by orotic acid was prevented with azaorotate. These studies provide substantial evidence that the inhibitory effect of orotic acid on purine synthesis in cultured human cells is due to a depletion of intracellular PP-ribose-P.

Glutamine Phosphoribosylpyrophosphate Amidotransferase

Publisher Summary Glutamate assays are compromised by the presence of glutaminase activity in crude preparations, but this is not a problem with purified preparations of amidotransferase. Assay of pyrophosphate is insensitive, but this assay has been used with addition of a basal level of inorganic pyrophosphate. Assay of phosphoribosylamine is complex and involves preparation of other enzymes. Only the spectrophotometric assay of glutamate with glutamate dehydrogenase and 3-acetylpyridine n icotinamide adenine dinucleotide (3-AcPyNAD) lends itself to continuous kinetic measurement of activity. It has been found that the initial phases of the PP-ribose-P amidotransferase reaction are non-linear when determined with enzymatic or radiochemical assays of glutamate in consecutive aliquots of the reaction mixture. The lag of the reaction reflects the conversion of inactive to active enzyme by PP-ribose-P and Mg 2+ .

Regulation of purine biosynthesis and turnover

The first specific precursor of purine biosynthesis de novo is β-5-phosphoribosyl-1-amine (PRA). There are no special regulatory mechanisms operating between PRA and IMP. Therefore, the amount of PRA produced per unit time will determine the rate of purine ribonucleotide biosynthesis de novo. PRA is synthesized from phosphoribosylpyrophosphate (PP-ribose-P) and glutamine, by amidophosphoribosyltransferase. The enzyme will also accept NH3, and isotopic studies in man suggest that the NH3 pathway may be quantitatively significant. Km values for PP-ribose-P range from 0.06 to 0.48 mm, for glutamine from 0.5 to 5.0 mm in various systems. Concentrations of PP-ribose-P in liver appear to be well below Km values, whereas those of glutamine are about equal to Km values of rat liver and human placental enzymes. Amidophosphoribosyltransferase is allosterically inhibited by purine ribonucleotide end products. Mixtures of 6-amino- and 6-hydroxypurine ribonucleotide inhibit synergistically. The human enzyme exists in two forms with MWs of 133,000 and 270,000. Purine ribonucleotides convert the small form to the large form; PP-ribose-P stabilizes the enzyme in the small form. In mixtures of the two forms, activity is proportional to the amount of the small form present. Changes in rates of purine biosyntheses de novo can be analyzed in terms of effects upon substrate levels of PP-ribose-P or glutamine, effects on amounts or activities of the amidophosphoribosyltransferase (including its sensitivity to end-products), or effects upon concentrations of inhibitory ribonucleotides. PP-ribose-P levels in mouse liver vary over a 2–10 fold range with feeding or administration of certain drugs. The highest values approach Km values of PP-ribose-P. Formation of PP-ribose-P from R5P and ATP is dependent upon allosteric activation of PP-ribose-P synthetase by Pi. The synthetase is inhibited by three mechanisms, involving 1) PP-ribose-P and 2,3-DPG, 2) ADP, 3) nucleotides in general by “heterogeneous metabolic pool inhibition,” but with relatively high Ki values.

Metabolic defects of primary hyperuricemia and gout

Abstract Gout is a syndrome of multiple pathogeneses rather than a single disease entity. Reviewed here are the metabolic defects of primary gout, with major emphasis upon two well characterized, although uncommon, variants due to specific enzyme abnormalities: (1) structural mutants of phosphoribosylpyrophosphate (PP-ribose-P) synthetase with increased activities, resulting in increased rates of synthesis of PP-ribose-P, a key substrate of purine biosynthesis, and (2) structural mutants of hypoxanthineguanine phosphoribosyltransferase (HGPRT) with reduced activities, resulting in reduced consumption of PP-ribose-P and therefore a surplus in the amount available for purine biosynthesis de novo. The present state of our limited knowledge of control of purine biosynthesis is also reviewed briefly, and the potential mechanisms of excessive uric acid production in idiopathic gout are discussed in terms of possible excesses of substrates (PP-ribose-P or L-glutamine) of the first specific reaction of purine biosynthesis, possible defects of control of the enzyme catalyzing this reaction, or defects in maintenance of optimal concentrations of nucleotide regulators of this reaction. It is likely that the rate of production of uric acid in man is influenced by availability of substrates, cofactors and regulatory compounds, and activities of enzymes at many reaction sites other than the first specific reaction of the purine sequence, but their influences upon the rate of purine production can be conveniently analyzed in terms of their indirect effects upon this reaction. Examples cited include glucose-6-phosphatase deficiency glycogen storage disease, in which marked hyperuricemia and purine overproduction are present, and elevated activities of hepatic xanthine oxidase in gouty patients with increased uricaciduria, perhaps occurring secondary to other factors but nevertheless contributing to the excessive purine production. The basic lesions of the more than 95 per cent of patients with primary gout who do not have abnormalities of either PP-ribose-P synthetase or HGPRT remain to be defined, but will almost certainly turn out to be multiple, complex and, in many cases, subtle deviations of metabolic control.

Genetic control of enzyme activity in higher organisms

The known intracellular controls of metabolism in higher organisms include regulation of transcription and translation, of enzyme turnover, and of enzyme activity. The magnitudes of changes of enzyme activity attributable to enzyme induction or catabolism in mammalian cells are small compared with those commonly observed during induction or repression in bacteria. Nevertheless, changes of five- to twenty-fold in activity in response to dietary changes or drug administration clearly are of metabolic significance, as, for example, in regulation of hepatic gluconeogenesis. Feedback controls provide a more versatile form of fast response for metabolic adaptations in cells of higher organisms and allowing tailoring of metabolism to exhaust or conserve different substrates intracellularly.

Human Glutamine Phosphoribosylpyrophosphate (PP-ribose-P) Amidotransferase: Kinetic, Regulation and Configurational Changes

In a large percentage of patients with gout, hyperuricemia is the result of an increase in the rate of purine biosynthesis de novo (Wyngaarden and Kelley, 1972). Consequently, it is important to understand the molecular basis for the regulation of purine biosynthesis in man.

Panel Discussion: Hyperuricemia as a Risk Factor

I wish to thank Professor Kaiser and Dr. Muller for having invited us to form this panel which is to discuss hyperuricaemia as a risk factor. I wrote to my colleagues about the four main topics which we are going to discuss today. We have to consider the extent to which hyperuricaemia may be a health risk factor, or an indicator of disease; whether it ever merits treatment in the absence of known complications, and if so, when and how it should be treated. We also have to identify the risks, or the alleged risks, and the importance of known or suspected pathophysiological factors, which may modulate the serum urate and urine uric acid concentrations in particular individuals. Under this heading, we have to think of such things as genetic, ethnic and dietary factors, sex and obesity.

A kinetic model for the intramolecular distribution of 15N in uric acid in patients with primary gout fed 15N-glycine

Abstract The concept of an abnormality of glutamine metabolism in primary gout was first proposed on the basis of isotope data: when 15 N-glycine was administered to gouty subjects, there was disproportionately great enrichment of N-(3+9) of uric acid, which derive from the amide-N of glutamine. An unduly high concentration of 15 N in glutamine was postulated, and attributed to a hypothetical defect in the catabolism of glutamine. Excess glutamine was proposed as the driving force of uric acid overproduction. We have reexamined this proposition in four gouty and three control subjects. In three of the gouty subjects the driving force of excessive purine biosynthesis was a known surplus of α-5-phosphoribosyl-1-pyrophosphate. The precursor glycine and glutamine pools were sampled by isolation of urinary hippurate and phenylacetylglutamine. Enrichment values of hippurate and of phenacetylglutamine were normal in all of the gouty subjects studied. However, the time course of 15 N enrichment of hippurate differed from that of the amide-N of glutamine. A kenetic model was constructed, and experimentally derived data were used to drive the model. It was found that disproportionate labeling of N-(3+9) could be produced from the model when appropriate constraints were used. Thus, preferential enrichment of N-(3+9) in gouty overproducers given 15 N-glycine does not necessarily reflect a specific abnormality of glutamine metabolism, but rather appears to be a kinetic phenomenon associated with accelerated purine biosynthesis per se.

REGULATION OF HUMAN GLUTAMINE PHOSPHORIBOSYLPYROPHOSPHATE AMIDOTRANSFERASE BY INTERCONVERSION OF TWO FORMS OF THE ENZYME

ABSTRACT. Human glutamine PP-ribose-P amidotransferase (PP-ribose-P amidotransferase) catalyzes the initial rate-limiting step in purine biosynthesis de novo . The catalytic activity of the enzyme is determined by the relative concentrations of purine ribonucleotides (feedback inhibitors) and PP-ribose-P (substrate). Two interconvertible forms of the enzyme with molecular weights of 133,000 and 270,000 have been identified. The small form of the enzyme is converted into the large form in the presence of purine ribonucleotides. The large form of the enzyme is converted into the small form in the presence of PP-ribose-P. Enzyme activity correlates directly with the amount of PP-ribose-P amidotransferase present in the small form of the enzyme. These studies provide a molecular basis for understanding the regulation of purine biosynthesis de novo by PP-ribose-P and purine ribonucleotides in man.

The Kinetics of Intramolecular Distribution of 15N in Uric Acid Following Administration of 15N-Glycine: Preferential Labeling of N−(3+9) of Uric Acid in Primary Gout and a Reappraisal of the “Glutamine Hypothesis”

Patients with primary gout and excessive uric acid excretion fed a test dose of 15N-glycine incorporate increased quantities of 15N into urinary urate [1–4]. Although enrichment of all 4 nitrogen atoms of uric acid is excessive [4,5] that of N−(3+9) is disproportionately great, especially in flamboyant overexcretors of uric acid [5]. Since N−3 and N−9 of uric acid are derived from the amide-N of glutamine [6,7], Gutman and Yu [5,8] proposed a defect in glutamine metabolism in primary gout. Since urinary ammonium, which arises principally from glutamine [9,10], is reduced in many gouty subjects [11,12], they further postulated a reduction of glutaminase activity [5,8]. The hyperglutamatemia of gout has now suggested a defect of glutamate metabolism, with diversion of glutamic acid toward glutamine and purine biosynthesis [13,14].