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Home , Eloise Giblett, Guanosine monophosphate, Inosinic acid, Purine, Purine metabolism

Ann Rheum Dis: first published as 10.1136/ard.42.Suppl_1.8 on 1 January 1983. Downloaded from

Ann Rheum Dis (1983), 42, Supplement p 8

Human : some recent advances and relationships with

GEORGE NUKI From the Rheumatic Diseases Unit, Department ofMedicine (WGH), University ofEdinburgh

Studies of human 0-6 g (3-6 mmol) while on a 10-9 MJ balanced interaction of several began some 200 years ago with (2600 kilocalorie), 70 g , biochemical pathways (Fig. 1). Purine Scheele's identification of as a purine-free diet is generally taken to de novo consists of a constituent of a renal calculus' and be abnormal,5 but recent data on series of 10 enzymatic steps in which Wollaston's demonstration of urate in healthy Australians on low purine , , dioxide, a tophus from his own ear.2 Just as diets showed mean urinary urates of aspartate, and 1-carbon-formyl these were among the earliest 3-9 mmol (0 65 g)/24 hrs for men and derivatives of are added to the experiments in the new science of 2-9 mmol (0.5 g)/24 hrs for women.6 moiety of phosphoribosyl , so Garrod's (1854)3 A variety of of three (PRPP) to form thread test must rank as the first assay are associated with excessive (IMP). in what we now call the discipline of purine synthesis in patients with , monophosphate (AMP) and clinical chemistry. Seegmiller's but these account for only a small monophosphate (GMP) are discovery, 15 years ago, that the severe proportion of those identified as each formed from IMP by two purine overproduction and gout primary overproducers of urate. enzymatic steps before phosphory- copyright. observed in boys with Lesch-Nyhan Purine synthesis in lation to their respective diphosphates syndrome resulted from a specific mammalian cells is regulated by a and triphosphates. Alternatively, the defect of the purine salvage - phospho- ribosyl transferase (HGPRT)4 led to Ribose - 5 - P + ATP what has been described as the 'purine revolution' and was soon followed by 5- Phosphoribosyl -1- Pyrophosphate PRPP + Glutamine Deoxynu~cetc Nuclic A * ,Nucleic Dexynucli~c the identification of other inborn Acids Acids ^ Acids Acids

errors of dGTPGT* * http://ard.bmj.com/ metabolism associated with dGTP GTP 5-Phosphoibosyl-1-,- AO dAAPAT P : primary purine overproduction and \\ \\\ F?1 ormate 1 /77 gout. Oxypurine and deoxypurine NHCO3 ' Aspartate bases, , and do, Formate however, have much wider physio- logical importance as intermediates in Guanylic Acd A-rIosindcAcid lo Adeyi Acid Adene d AMP energy metabolism, components of HGPRT ne coenzymes, vasoactive and neuro- Guanosie Io eR Adenosone N transmitter substances, mediators of 9f % 1 > 2,8-dihydroxyademne on September 27, 2021 by guest. Protected hormonal action, and as the building Guanine Hypoxanthine deoxyinosre -i' dexyaadenoswe blocks for transmitters of genetic information (DNA and RNA). Most recently interest has been focused on the role of , and especially deoxypurines, in the regulation of Uric cid immune responses. Fig. 1 Pathways ofpurine metabolism in man. Gout and hyperuricaemia ADA = . APRT = phosphoribosyl transferase. In most individuals with gout or HGPRT = Hypoxanthine-guanine phosphoribosyl transferase. asymptomatic hyperuricaemia the rise NP = phosphorylase. in serum urate concentration is the 5'NT = 5'. result of a decrease in renal urate PAT = Phosphoribosyl pyrophosphate amidotransferase. clearance. In 15% or less there is PPRPS = Phosphoribosyl pyrophosphate synthetase. evidence of excessive purine synthesis XO = . and . A 24-hour Reproduced from: Nuki G, Advanced Medicine 15. London: Pitman Medical, urinary urate excretion greater than 1979: 140 with permission of publishers. Ann Rheum Dis: first published as 10.1136/ard.42.Suppl_1.8 on 1 January 1983. Downloaded from

Human purine metabolism: some recent advances Suppl p 9 nucleotide monophosphates may be the potential regulatory importance of metabolites from uric acid." These synthesised by phosphorylation of the distal inosinic acid branch point." patients may be treated successfully purine bases by phosphoribosyl trans- There is increasing evidence of with a low purine diet and ferase enzymes reacting with PRPP, genetic heterogeneity at the HGPRT ," which inhibits the and AMP may be formed by locus."2 13 Although the severity of formation of 2,8-dihydroxyadenine. phosphorylation of adenosine by clinical manifestations is usually adenosine kinase. In vitro studies of proportional to the severity of the Superactive PRPP synthetase amido phosphoribosyl transferase enzyme defect, this is not always (PAT), the first enzyme in the de novo apparently the case. In rare instances Structural mutations resulting in pathway, show it to have allosteric mutations resulting in abnormal superactive phosphoribosyl pyro- properties that one often associates enzyme kinetics may be associated on synthetase are now well with a rate-limiting enzyme, and have the one hand with the classic clinical established as a cause of X-linked gout provided a molecular model for the Lesch-Nyhan phenotype and and purine overproduction. Initially mechanism whereby the rate of purine apparently normal red HGPRT increased enzyme activity was associ- nucleotide synthesis may be regulated. activity assayed at saturating substrate ated with decreased sensitivity to The protein exists in two forms; an concentrations, or on the other hand feedback inhibition by purine nucleo- inactive dimer and a catalytically with X-linked gout without tides in one family" and a primary active monomer. PRPP converts the neurological features and apparently increase in catalytic activity per larger form of the enzyme to the active absent enzyme activity in standard enzyme of the mutant pro- monomer, while purine nucleotide assays. Immunochemical studies have tein in another.'4 More recently the monophosphates have the reverse shown that most mutations at the genetic heterogeneity and diversity of effect.7 HGPRT locus are structural gene abnormalities in kinetic mechanisms mutations resulting in catalytically leading to superactivity of this enzyme HGPRT deficiency defective enzyme protein that does not has been extended to include mutants cross react with antibody raised with increased V max, various abnor- In Lesch-Nyhan syndrome and the against highly purified normal human malities of purine nucleotide feedback severe familial form of X-linked gout, HGPRT (CRM negative mutants).'4 inhibitor responsiveness, increased copyright. which is also associated with deficiency Recent studies have provided affinity for the substrate ribose-5- of HGPRT, it seems logical to evidence for a single phosphate, and a combination of conclude that the considerably substitution in one such structural catalytic and regulatory defects." accelerated rate of de novo purine variant (HGPRTmunich)," while other Most affected males have presented synthesis results from both groups have now succeeded in cloning with gouty arthritis and/or uric acid accumulation of PRPP and diminished the HGPRT gene.' urolithiasis in early adult , but two feedback inhibition of PAT by purine families have been described where nucleotides. However, while APRT deficiency homozygous boys developed symp- toms in childhood and shared

intracellular concentrations of PRPP http://ard.bmj.com/ are increased in HGPRT-deficient Adenine phosphoribosyl transferase metabolic abnormalities and nerve cells, steady state purine nucleotide deficiency is not associated with gout deafness with their heterozygous concentrations do not appear to be or purine overproduction. Although mothers.'6 27 reduced,8 and concentrations of heterozygous, partial APRT nucleotides are actually deficiency was originally described in Type I glycogen storage disease increased.9 A good deal of evidence gouty patients it is clear from family suggests that PRPP, which is present in studies that the enzyme defect and Glucose-6-phosphatase deficiency is cells at limiting concentrations, is the hyperuricaemia are inherited as associated with both overproduction major regulator of rates of de novo independent traits.'8 The apparent and underexcretion of uric acid, so that on September 27, 2021 by guest. Protected purine synthesis whenever purine association arose spuriously because gout and hyperuricaemia become overproduction occurs in vivo. On the APRT enzyme assays were appreciable clinical problems in other hand, the importance of purine undertaken only in patients with gout, affected individuals fortunate enough base 'salvage' is emphasised by although heterozygous APRT to survive to adult life. Persistent lactic experiments which show that rates of deficiency may occur in as many as 1 % acidaemia is the major cause of purine synthesis in normal human of the population.'9 More recently, a impairment of uric acid excretion and lymphoblasts may be increased to number of patients have been the simultaneous increase in de novo those observed in HGPRT-deficient identified in whom severe, purine synthesis observed in these mutants if hypoxanthine is carefully homozygous APRT deficiency was patients is thought to result from excluded from the culture medium."0 associated with symptomatic urinary excessive production of PRPP after Moreover, Hershfield has lithiasis and calculi composed of 2, 'shunting' of metabolites through the demonstrated co-ordinated inhibition 8-dihydroxyadenine.20 21 X-ray phosphate pathway.'8 It has of IMP conversion to adenine diffraction, infrared and ultraviolet been difficult to test this hypothesis nucleotides with enhancement of total absorption spectrometry, and ion direct, as glucose-6-phosphatase guanine nucleotide synthesis after the exchange chromatography of HPLC activity is limited to the , , addition of adenosine to human extracts of these stones have been and intestinal mucosa. Recently it has lymphoblasts in culture, emphasising required to differentiate the adenine been suggested that increased purine Ann Rheum Dis: first published as 10.1136/ard.42.Suppl_1.8 on 1 January 1983. Downloaded from

Suppl p 10 Annals ofthe Rheumatic Diseases production in this disorder may be years later also reported and there is no evidence to secondary to accelerated purine a gross deficiency of red cell purine suggest that it is causally related to degradation after recurrent nucleoside phosphorylase (PNP) in B-cell deficiency. 5'NT deficiency in and depletion of children with recurrent infections and X-linked agammaglobulinaemia ATP29; rather like the mechanism a predominant T-lymphocyte largely reflects the deficit in circulating accounting for the purine deficiency.36 B-cells which have high 5'NT activity. overproduction and hyperuricaemia The causal relationship between B-cell numbers are, however, almost which follow ingestion or infusion of these inherited enzyme abnormalities normal in common variable hypo- fructose.30 Van den Berghe and his and the immunodeficiency syndromes gammaglobulinaemia and in this situa- colleagues believe that the hepatic is now established and has highlighted tion it seems likely that reduction in of adenine nucleotides and the need for an intact purine catabolic 5'NT is associated with a population of the formation of uric acid are pathway to maintain normal cellular relatively immature B-lymphocytes. regulated by the activity of AMP and humoral immunity. In patients 5'NT has been previously shown to be deaminase.3' with deficiencies of ADA and PNP a marker of differentiation in both T46 Type I glycogen storage disease is plasma concentrations of deoxy- and B47 lymphocytes. usually characterised by complete adenosine37 and deoxyguanosine3" absence of hepatic glucose-6- are raised. These deoxynucleosides References phosphatase. We have recently are selectively phosphorylated and reported the case of a child with a par- trapped by T-lymphocytes with high 1 Scheele K W. Examen chemicum calculi tial enzyme deficiency where there was activities of kinase and urinarii. Opuscula 1976; 73: 2. 2 Wollaston W H. On gouty and urinary a striking absence of hypoglycaemia.32 low activities of intracellular deoxy- concretions. Philos Trans R Soc Lond This suggests that type I glycogen stor- nucleotidase. As a result deoxy- 1797; 87: 386. age disease with partial deficiency of adenosine39 and dexoyguanosine4 3 Garrod A B. On the blood and effused hepatic glucose-6-phosphatase should accumulate in the cells, leading to fluids in gout, rheumatism and Bright's perhaps be considered in patients with inhibition of reductase Disease. Transactions of the gout or hyperuricaemia associated and DNA synthesis. An alternative Medico-Chirugical Society ofEdinburgh 1854; 37: 49. with hypertriglyceridaemia and lactic mechanism for the toxicity of deoxy- copyright. acidaemia, even in the absence of adenosine in ADA deficiency has 4 Seegmiller J E, Rosenbloom F M, Kelley W N. Enzyme defect associated hypoglycaemia. been proposed by Hershfield,4" who with a sex-linked neurological disorder showed that it causes irreversible 'sui- and excessive purine synthesis. Science Other enzyme defects and gout cide' inactivation of the enzyme 1967; 155: 1682-4. are still unable to define the S-adenosyl homocysteine hydrolase. 5 Seegmiller J E, Grayzel A I, Laster L, We Consistent with this hypothesis Liddle L. Uric acid production in gout.J metabolic abnormality responsible for Clin Invest 1961; 40: 1304-14. in most of the S-adenosyl homocysteine hydrolase purine overproduction activity is found to be considerably 6 Stafford W, Emmerson B T. Effect of few patients with gout who are primary reduced in the red cells of children purine restriction on serum and urinary hyperexcretors of uric acid. Though urate (Abstract). J Clin Chem Clin be attributable with ADA deficiency,42 but an alterna- Biochem 1982; 20: 422-3. http://ard.bmj.com/ some of these may well tive to to subtle kinetic variations of known explanation needs to be found 7 Holmes E W, Wyngaarden J B, in explain the T-cell deficiency in PNP Kelley W N. Human glutamine, phos- enzyme defects not detectable deficiency, as does standard in vitro assays, it seems phoribosyl pyrophosphate amido trans- that not inhibit the S-adenosyl homocys- ferase: two molecular forms inter- probable other primary purine teine hydrolase enzyme. convertible by purine enzyme defects remain to be and phosphoribosyl pyrophosphate. J discovered. For example, in vivo33 and To explain the difference in the Biol Chem 1973; 248: 6035-40. in vitro34 studies with drugs that inhibit immunodeficiency syndromes 8 Rosenbloom F M, Henderson J F, IMP dehydrogenase suggest that associated with ADA and PNP defi- Caldwell I C, Kelley W N, Seeg- on September 27, 2021 by guest. Protected deficiency ofthis enzyme might well be ciency, evidence has recently been miller J E. Biochemical bases of accele- associated with primary purine produced to show that intracellular rated purine biosynthesis de novo in overproduction. ATP activities are depleted with human fibroblasts lacking hypo- accumulation of but xanthine-guanine phosphoribosyl trans- not with deoxyguanosine, so killing the ferase. J Biol Chem 1968; 243: PURINE ENZYME 1166-73. ABNORMALITIES AND non-dividing helper T-lymphocytes.43 9 Nuki G, Astrin K, Brenton D, IMMUNODEFICIENCY Humoral immunity persists in patients Cruikshank M, Lever L, Seegmiller J E. A great deal of interest has been with PNP deficiency because T-hel- Purine and pyrimidine nucleotide recently aroused by the association of per function is relatively proliferation concentrations in cells with decreased two inborn errors of purine independent. hypoxanthine-guanine phosphoribosyl metabolism with immune deficiency Decreased activity of the ectopurine transferase (HGPRT) activity.Adv Exp 5' nucleotidase enzyme has Med Biol 1977; 76A: 326-9. syndromes. (5'NT) 10 Hershfield M S, Seegmiller J E. Severe deficiency of adenosine been associated with X-linked"4 and Regulation of de novo purine synthesis deaminase (ADA) was first reported acquired adult onset (common vari- in human lymphoblasts: similar rates of in red cell haemolysates from two able) hypergammaglobulinaemia.45 during growth by children with severe combined Deficiency of this enzyme does not, normal cells and mutants deficient in immunodeficiency in 1972.3 Three however, result from a primary gene hypoxanthine-guanine phosphoribosyl Ann Rheum Dis: first published as 10.1136/ard.42.Suppl_1.8 on 1 January 1983. Downloaded from

Human purine metabolism: some recent advances Suppl p 11

transferase activity. J Biol Chem 1977; phosphoribosyl transferase deficiency: Sandiman R, Diamond L K. Nucleoside 252: 6002-10. effect of diet and allopurinol therapy. phosphorylase deficiency in a child with 11 Hershfield M S, Seegmiller J E. Adv Exp Med Biol 1977; 76B: 304-11. severely defective T-cell immunity and Regulation of de novo purine 23 Sperling Q, Persky-Brosh S, Boer P, De normal B-cell immunity. Lancet 1975; biosynthesis in human lymphoblasts: Vries A. Human erythrocyte phospho- i: 1010-3. co-ordinate control of proximal rate ribosyl pyrophosphate synthetase muta- 37 Simmonds H A, Panayi G S, Corigal V. determining steps and the inosinic acid tionally altered in relatory properties. A role for purine metabolism in the branch point. J Biol Chem 1976; 251: Biochem Med 1973; 7: 389-95. immune response, adenosine deaminase 7348-54. 24 Becker M A, Kostel P J, Meyer L J, activity and deoxyadenosine 12 Emmerson B T, Thompson L. The Seegmiller J E. Human phosphoribosyl catabolism. Lancet 1978; i: 60-3. spectrum of hypoxanthine-guanine pyrophosphate synthetase: increased 38 Cohen A, Doyle D, Martin D W Jr, phosphoribosyl transferase deficiency. enzyme activity in a family with gout and Ammann A J. Abnormal purine Q J Med 1973; 42: 423-40. excessive purine synthesis. Proc Natl metabolism and purine over-production Acad Sci USA 1973; 70: 2749. in a patient deficient in purine 13 Nyhan W L. Genetic heterogeneity at 25 Becker M A. Phosphoribosyl pyro- nucleoside phosphorylase. N EngI J the locus for hypoxanthine-guanine phosphate synthetase superactivity: Med 1976; 295: 1449-54. phosphoribosyl transferase. In: Purine detection, characterization of underly- 39 Donofino J, Coleman M S, Hutton J J, and pyrimidine metabolism, Ciba ing defects and treatment (Abstract). J Duoud A, Lomplein B, Difminski J. Foundation Symposium No 48. Clin Chem Clin Biochem 1982; 20: 347. Over production of adenine Amsterdam. Excerpta Medica: 65-81. 26 Becker M A, Raivio K 0, Bakay B, deoxynucleosides and deoxynucleotides 14 Upchurch K S, Leyva A, Arnold W J, Adams W B. Variant human phos- in adenosine deaminase deficiency with Holmes E W, Kelley W N. Hypo- phoribosyl pyrophosphate synthetase severe combined immunodeficiency xanthine phosphoribosyl transferase altered in regulatory and catalytic func- disease. J Clin Invest 1978; 62: 884. deficiency: association of reduced tions. J Clin Invest 1980; 65: 109-20. 40 Cohen A, Goudas L J, Ammann A J, catalytic activity with reduced levels of 27 Simmonds H A, Webster D R, Wilson J, Staal G E J, Martin D W Jr. immunologically detectable enzyme Lingham S. An X-linked syndrome Deoxyguanosine tri-phosphate as a protein. Proc Natl Acad Sci USA 1975; characterised by hyperuricaemia, deaf- possible toxic metabolite in purine 72: 4142-6. ness and neurodevelopmental abnor- nucleoside phosphorylase deficiency. J 15 Wilson J M, Tait G E, Grobner W, Zol- malities. Lancet 1982; fi: 68-70. Clin Invest 1978; 61: 1405. lner N, Kelley W N. Evidence for an 41 Hershfield M S. Suicide inactivation of 28 Howell R R. The interrelationship of copyright. amino acid substitution (Ser Arg) in the colycogen storage disease and gout. human lymphoblast S-adenosyl hypoxanthine-guanine phosphoribosyl Arthritis Rheum 1965; 8: 780-5. homocysteine hydrolase by transferase variant HGPRTmunnCh (Abs- 29 Greene H C, Wilson F A, Hefferan P, et 2'-deoxyadenosine and adenine tract). J Clin Chem Clin Biochem 1982; al. ATP depletion, a possible role in the arabinoside: a basis for direct toxic 16 Jolly D J, Esty A, Friedmann T. The pathogenesis of hyperuricaemia in effects of analogs of adenosine. J Biol cloned human hypoxanthine-guanine glycogen storage disease type I. J Clin Chem 1979; 254: 22. phosphoribosyl transferase gene Invest 1978; 62: 321-8. 42 Hershfield M S, Knedich N M, Ownby D R, Ownby H, Buckley R J. In (Abstract). J Clin Chem Clin Biochem 30 Raivio K 0, Becker M A, Meyer L J, 1982; 20: 380. Greene M L, Nuki G, Seegmiller J E. vivo inactivation of erythrocyte 17 Chinault A C, Konecki D S, Brennand J, Effects of fructose infusion on urate S-adenosyl homocysteine hydrolase by 2'-deoxyadenosine in adenosine Patel P, Melton D W, Caskey C T. production in man. Metabolism 1975; http://ard.bmj.com/ Cloning and characterisations of 24: 861-9. deaminase deficient patients. J Clin mammalian hypoxanthine-guanine 31 Van den Berghe G, Bronfman M, Invest 1979; 63: 807. phosphoribosyl transferase genes Vanneste R, Hers H G. The mechanism 43 Carson D A, Wasson D B, Lakow E, (Abstract). J Clin Chem Clin Biochem of depletion in Kamatami M. Biochemical basis for 1982; 20: 442. the liver after a load of fructose. lymphocyte dysfunction in adenosine 18 Emmerson B T, Gordon R B, Biochem J 1977; 162: 601-9. deaminase and purine nucleoside Thompson L. Adenine phosphoribosyl 32 Nuki G, Parker J. Clinical and phosphorylase deficiencies.J Clin Chem transferase deficiency in a female with enzymological studies in a child with Clin Biochem 1982; 20: 355. Med Biol 41: type I glycogen storage disease 44 Edwards M L, Magilavy D B, Cas-

gout. Adv Exp 1974; on September 27, 2021 by guest. Protected 327-31. associated with partial deficiency of sidy J T, Fox I H. Lymphocyte ecto-5'- 19 Fox I H, Lacroix S, Planet G, Moore N. hepatic glucose-6-phosphatase. Adv nucleotidase deficiency in agammag- Partial deficiency of adenine Exp Med Biol 1979; 122A: 189-202. lobulinaemia. Science 1978; 201: phosphoribosyl transferase in man. 33 Seegmiller J E, Grayzel A I, Liddle L, 628-30. Medicine 1977; 56: 515-26. Wyngaarden J B. The effect of 45 Johnson S M, North M E, Asher- 20 Cartier M B, Hanset M. Une nouvelle 2-ethyl-amino-1,3,4-thiadizole on the son G L, Allsop J, Watts R W E, maladie metabolique: le deficit complex incorporation of glycine into urinary Webster A D B. Lymphocyte purine en adenine phosphoribosyl transferase purines and uric acid in man. 5'-nucleotidase deficiency in primary avec lithiase de 2, 8-dihydroxyadenine. Metabolism 1963; 12: 507-15. hypogammaglobulins. Lancet 1977; i: Comptes Rendus Hebdomadaires des 34 Willis R C, Carson D A, Seegmiller J E. 168-70. Seances de lAcademie des Sciences Adenosine kinase initiates the major 46 Edwards M L, Gelfund E W, Burk L, (Paris) 1974; 270: 883-6. route ofribavirin activation in a cultured Dosch H M, Fox I H. Distribution of 5' 21 Simmonds H A, Van Acker K J, human cell line. Proc NatlAcad Sci USA nucloeotidase in human lymphoid Cameron J S, Sneddon W. The 1978; 75: 3042-4. tissues. Proc Natl Acad Sci USA 1979; identification of 2, 8-dihydroxyadenine, 35 Giblett E R, Anderson J E, Cohen F, 76: 3474-6. a new component of urinary stones. Pollara B, Menwissen H J. Adenosine 47 Boss G R, Thompson L F, Spregel- Biochem J 1976; 157: 485-7. deaminase deficiency in two patients heng H L, et al. Lymphocyte ecto-5'- 22 Simmonds H A, Van Acker K J, with severely impaired cellular nucleotidase activity as a marker of Cameron J S, McBurney A. Purine immunity. Lancet 1972; ii: 1067-9. B-cell maturation. Trans Assoc Am excretion in complete adenine 36 Giblett E R, Ammann A J, Warn D W, Physicians 1979; 92: 309-15.