Nfletffillflsm of Nuelieotfl dles

ucleotides \f consistof a nitrogenousbase, a | \ pentose and a phosphate. The pentose sugaris D-ribosein ribonucleotidesof RNAwhile in deoxyribonucleotides(deoxynucleotides) of i Aspariaie--'N.,,,t .J . DNA, the sugaris 2-deoxyD-ribose. Nucleotides t participate in almost all the biochemical processes/either directly or indirectly.They are the structuralcomponents of nucleicacids (DNA, Y RNA), coenzymes, and are involved in tne Glutamine regulationof severalmetabolic reactions. Fig. 17.1 : The sources of individuat atoms in ring. (Note : Same colours are used in the syntheticpathway Fig. lZ.2).

n T. C4, C5 and N7 are contributedby glycine. Many compoundscontribute to the purine ring of the nucleotides(Fig.t7.l). 5. C6 directly comes from COr. 1. purine N1 of is derivedfrom amino group It should be rememberedthat purine bases of aspartate. are not synthesizedas such,but they are formed as ribonucleotides. The 2. C2 and Cs arise from formate of N10- are built upon a formyl THF. pre-existing ribose S-phosphate. Liver is the major site for purine nucleotide synthesis. 3. N3 and N9 are obtainedfrom amide group Erythrocytes,polymorphonuclear leukocytes and of glutamine. brain cannot producepurines. 388 BIOCHEMISTF|Y

m-gg-o-=_ |l Formylglycinamide ribosyl S-phosphate Kn H) Glutam H \-Y OH +ATt OH OH Glutame cl-D-Ribose-S-phosphate + ADP orr-l t'1 PRPPsYnthetase ,N o"t*'] \cH + Hrcl-itl HN:C-- O EO-qn2-O.- H -NH l./ \l KH H) I u \.]_j^/ r,\-iEl-/^\-td Ribose5-P II Formylglycinamidineribosyl-s-phosphate OH OH I S-Phosphoribosylo-pyrophosphate ATP\l ) Synthetase ADP+ PiYl PRpp glutamyl amidotrahsfera-se +H2O +

E-o-gH4.o-\ NHz 1../ \ | H) \H I HtsrH Ribose5-P OH OH S-Amlnoimidazoleribosyl-S-phosphate p-S-Phosphoribosylamine I aor--l ,, I Carborytase ;;,.:i!:r+ ATp_.rl I I Synthetaee I ADP+ Pia'l o+ + tl CH2 f.iHZ ;-; -/-/ E-o-cHz-o.- NH D/ \l f\H H) H\-Jn Ribose5-P tt 5-Aminolmidazolecarborylate OH OH rlbosyl s-phosphate Glycinamiderlbosyl S-phosphate Aspartate+Al

rrmyltransferase ADP+I

n -ooc li | H -51 f.l HC- ir, l\_ .PH t,, --'\cH CH, r- / ,.1::r.. il co( O I NH Ribose5-P I Ribose S-Aminoimidazole 5-p 4-succinylcarboxamide Formylglycinamideribosyl S-phosphate ribosyl 5-phosphate

Fag. l?.2 contd. next column Fag. 17.2 contd. next page Ghapter 17 : METABOLISMOF NUCLEOTIDES 389

5-Aminoimidazole 1. Ribose 5-phosphate, produced in the 4-succinylcarboxamide ribosyl 5-phosphate hexose monophosphateshunt of carbohydrate metabolism is the starting material for purine nucleotidesynthesis. lt reactswith ATP to form phosphoribosylpyrophosphate (PRPP).

2. Clutamine transfersits amide nitrogento ll PRPP to replace pyrophosphateand produce 5-phosphoribosylamine.The PRPP glutamyl amidotransferase is controlled by feedback inhibition of nucleotides (lMP, AMP and GMP). This reaction is the 'committed step' Ribose5-P in purine nucleotidebiosynthesis. S-Aminoimidazole 4-carboramide ribosyl S-phosphate 3. Phosphoribosylaminereacts with glycine in the presenceof ATP to form glycinamide ribosyl 5-phosphate or glycinamide ribotide (cAR).

4. N10-Formyltetrahydrofolate donates the group tl formyl and the formed is formyl- glycinamideribosyl 5-phosphate.

5. Clutamine transfers the second amido amino group to produce formylglycinamidine ribosyl 5-phosphate.

5-Formaminoimidazole 6. The imidazole ring of the purine is closed 4-carboxamide yield ribosyl 5-phosphate in an ATP dependentreaction to 5-amino- I imidazole ribosyl S-phosphate. I u n) Cyclohydrolase 7. Incorporation of COz (carboxylation) + occurs to yield aminoimidazole carboxylate o ribosyl 5-phosphate. This reaction does not tl require the vitamin biotin and/or ATP which is the case with most of the carboxvlation reactions.

8. Aspartatecondenses with the product in Ribose5-P reaction 7 to form aminoimidazole4-succinvl lnoaane monophosphate carboxamideribosyl S-phosphate.

Adenosuccinatelyase fumarate Fig. 17.2 : The metabolicpathway for the 9. cleavesoff synthesisof inosine monophosphate,the parent purine and only the amino group of aspartateis retained n ucleotide (P R PP-Phosphori bosyl pytophosph ate ; to yield aminoimidazole4-carboxamide ribosyl PPi-Pyrophosphate). 5-phosphate.

10. N1O-Formyltetrahydrofolate donates a The pathway for the synthesis of inosine one-carbon moiety to produce formamino- monophosphafe (lMP or inosinic acid), the imidazole 4-carboxamide ribosyl 5-phosphate. 'parent' purine nucleotide is given in Fig.l7.2. With this reaction,all the carbon and The reactionsare brieflv described in the next atoms of purine ring are contributed by the column. resoectivesources. 390 BIOCHEMISTFIY

reaction o 1'l. The final tl catalysed by cyclo- Hr.r/-YN\ leads to ring |il) closurewith an elimination \rlT, of water molecule. The Ribose5-P productobtained is inosine InosinemonophosPhate (lMP) monophosphate(lMP), the Asoartate+ GTP parent purine nucleotide IMP dehydrogenase from which other purine NAD: GDP+ Pi r HrO nucleotidescan be synthe- Adenylsuccinate synthetase sized. NADH + H* rcoc-cH2-?H-coo- o lnhibitors of NH purine synthesis I Folic acid (THF) is ru---YNr\ essentialfor the synthesis |il) of purine nucleotides \*tT, XanthosinemonophosPhate (reactions 4 and 10). Ribose5-P (XMP) Sulfonamides are the Adenylsuccinate Glutamine structuralanalogs of para- + ATP+ HrO I aminobenzoic acid Adenylsuccinase Fumarate4 (PABA).These sulfa drugs I Glutamate+ can be used to inhibit the + AMP + PPi synthesis of folic acid by NHz o microorganisms. This N. the \\ indirectlv reduces ) synthesisof purines and, / H2 N' therefore,the nucleicacids I Ribose5-P (DNA and RNA). AdencinemonophcPhab GuanosinemonoPhosPhate Sulfonamides have no (AMP) (GMP) influence on humans, acid is not since folic Fig. 17.3 : Synthesisof AMP and GMP from inosine monophosphate. svnthesized and is suppliedthrough diet. The structural analogs of folic acid (e.9. (Fig.l7.3).Aspartate condenses with IMP in the methotrexate) are widely used to control cancer. presence of CTP to produce adenylsuccinate They inhibit the synthesisof purine nucleotides which, on cleavage,forms AMP. (reaction4 and 10)and, thus, nucleic acids. Both For the synthesisof CMP, IMP undergoes these reactionsare concernedwith the transferof NAD+ dependent dehydrogenation to form one-carbon moiety (formyl group). These xanthosine monophosphate(XMP). Glutamine inhibitorsalso affectthe proliferationof normally then transfersamide nitrogento XMP to produce growing cells. This causes many side-effects CMP. includinganemia, baldness, scaly skin etc. 6-Mercaptopurineis an inhibitor of the of AMF $yrrthesis synthesis of AMP and GMP. lt acts on and GMP from IMP the enzyme adenylsuccinase (of AMP lnosine monophosphatets the immediate pathway) and IMP dehydrogenase(of GMP precursorfor the formationof AMP and GMP pathway). Shapt*rr 17 : METABOLTSM OF NUCLEOTTDES 391

Nucleosidemonophosphate the metabolicreactions. (AMP,cMP) This is achievedby the I transferof phosphategroup from ATp, ATP\l catalvsed by nucleosidemonophosphate (NMp) NMPkinase kinases ,l and nucleoside diphosphate (NDp) kinases ADPT- (Fig Yi 17.4). Nucleosidediphosphate (ADP,cDP) Salvage E**'B0?wayfor p*rfine*s I ATP\l The free purines (adenine, guanine and NDpkinase J hypoxanthine)are formed in the normalturnover ADP*- I + of nucleic acids (particularlyRNA), and arso Nucleotidetriphosphate obtained from the dietary sources.The purines (ATE GTP) can directly _be converted to the corresponding nucleotides, Fig" 17.4 : Conversionof nucteosidemonophosphates and this process is known as to di- and triphosphates(NMp-Nucleoside mono- 'salvage pathway' (Fig.l 7.fl. phosphate; NDP-Nucleosjde diphosphate). Adenine phosphoribosyltransferase catalyses the formation of AMp from adenine. Forrnatiem of p*erime n*86;Ee#side Hypoxanthine-guaninephosphoribosyl trans_ ferase (HCPRT) riipFros;phaBes em# trfrea[ao*pfu ote* converts guanine ano hypoxanthine,respectively, to CMp and lMp. The nucleosidemonophosphates (AMp ano Phosphoribosylpyrophosphate (pRpp) is rne CMP) haveto be converted to the corresponding donor of ribose 5-phosphate in the salvaee di- and triphosphatesto participatein most of pathway.

Adeninephosphoribosyl- Iransterase

Adenine 4p1p Ribose5-P oo Hypoxanthine-guanine H^r/^\yN\"'l' ^, * t'),n,-/---N., II \ pnosphoribosyltransferase+ tf \ l- tt / Hlr\*A^rl ffi rr*\*Ar) Guanine GMP Ribose5-p

Fig' 17-5 : Salvagepathways of purine nucteotide synthesis(PRPP-phosphoribosyt pyrophosphate; PPi-lnorganic pyrophosphate; AMP-Adenosine monophosphate: GMp-Guanosine monophosphate; IMP-lnosinemonophosphate; * Detieiencyof HGpRT causesLesch-Nyhan "Vni[oiJ". 392 BIOCHEMISTF|Y

Base O-o-O-o-H2c //'o\Base RibonucreotidereducraseO-o-O-o-t,? r,'o-

ry-juffiOH OH fi;).OHH Ribonucleoside Thioredoxin Thioredoxin Ribonucleoside diphosphate (2SH,reduced) (S-S-, oxidized) diphosphate (ADP,GDP, (ADP,GDP, CDEUDP) cDe UDP)

NADP* NADPH+ H*

Fig. 17.6 : Formation of deoxyribonucleotides frcm ribonucleotides.

The salvagepathway is particularlyimportant moiety (Fig.t7.6).This reactionis catalysedby a in certaintissues such as erythrocytesand brain multisubunit (two B1 and two 82 subunits) where de novo (a new) synthesisof purine enzvme, rihonucleotide reductase. nucleotidesis not operative. Supply of reducing equivalents : The enzyme A defect in the enzyme HGPRTcauses Lesch' ribonucleotide reductase itself provides the Nyhan syndrome (details given later). hydrogen atoms needed for reduction from its sulfhydrylBroups. The reducingequivalents, in Regulation of purine turn, are supplied by thioredoxin, a monomeric nucleotide biosynthesis protein with two cysteineresidues.

The ourine nucleotide synthesis is well NADPH-dependent thioredoxin reductase coordinatedto meet the cellular demands.The converts the oxidized thioredoxin to reduced intracellular concentration of PRPP regulates form which can be recycled again and again' purine synthesisto a large extent.This, in turn, Thioredoxin thus serves as a protein in is dependent on the availability of ribose an enzymatic reaction. S-phosphateand the enzyme PRPPsynthetase. Regulationof deoxyribonucleotide synthesis: PRPPglutamyl amidotransferaseis controlled Deoxyribonucleotidesare mostlyrequired for the bv a feedback mechanismby purine nucleotides. synthesisof DNA. The activity of the enzyme That is, if AMP and CMP are available in ribonucleotidereductase maintains the adequate adequate amounts to meet the cellular supply of deoxyribonucleotides. requirements,their synthesisis turned off at the is a complex amidotransferase reaction. Ribonucleotide reductase enzyme with multiple sites ( and is in the Another importantstage of regulation allosteric sites) that control the formation of AMP conversion of IMP to AMP and CMP. deoxvribonucleotides. inhibits adenylsuccinatesynthetase while CMP inhibits IMP dehydrogenase.Thus, AMP and CMP control their respectivesynthesis from IMP by a feedbackmechanism.

Conversion of ribonucleotides to deoxyribonucleotides The end product of in The synthesis of purine and pyrimidine humans is . fhe sequence of reactions deoxyribonucleotides occurs from ribo- in purine nucleotide degradation is given in nucleotidesby a reduction at the Cr of ribose Fig.17.7. ffihapter "T7; METABOLTSMOF NUCLEOTTDES 393

AMp deaminase AMp,- I H2u-..1.. H,o ilt. Hzor . J Nucleotidase Yt{^. +| Pir o

Ribose Adenosine lno

Pi_. Ribose a_ l-phosphate

n Hry--,\ tl Lr \-N/ Hypoxr

H2O + O2:,

H2o2y' I n o

,,,u H Xanthine Guanine

Xanthine oxidase

o

Uric acid

Fig. 17.7: Degradationof purine nucteotidesto uricacid (AMp_Aa"nii*iffiipnrr", lMp-lnosine monophosphate;GMp_Guanosne monophasphate). 394 BIOCHEMISTFIY

Uricacid Most animals (other than primates)however, I un""r" oxidize uric acid by the enzyme uricase to J allantoin, where the purine ring is cleaved. Allantoin Allantoin is then convertedto allantoicacid and I I Allantoinase excreted in some fishes (Fig.|7.8). Further + degradation of allantoic acid may occur to Allantoicacid produce (in ,most fishesand I Allantoicase (in Glvoxvlate+_,,1 some molluscs) and, later, to + marine invertebrates). Urea I I J Ammonia

Fig. 17.8 : Degradation of utic acid in animals other than man. Hyperuricemia and gout

1. The nucleotide monophosphates(AMP, Uric acid is the end product of purine IMP and CMP) are convertedto their respective metabolism in humans. The normal nucleoside forms (adenosine, inosine and concentrationof uric acid in the serumof adults guanosine) by the action of nucleotidase. is in the range of 3-7 m{dl. In women, it is slightlylower (by about 1 mg) than in men. The 2. fhe amino group, either from AMP or daily excretionof uric acid is about 500-700 mg. adenosine,can be removed to produce IMP or inosine,respectively. Hyperuricemia refers to an elevation in the serum uric acid concentration.This is sometimes guanosine respectively, 3. Inosine and are/ associatedwith increased uric acid excretion convertedto hypoxanthineand guanine (purine (uricosuria). bases) by purine nucleoside phosphorylase. Adenosine is not degraded by this enzyme, Gout is a metabolic disease associatedwith hence it has to be convertedto inosine. overproduction of uric acid. At the physiological pH, uric acid is found in a more solubleform as 4. Cuanine undergoes deamination by sodium urate. ln severehyperuricemia, crystals guanaseto form xanthine. of sodium urateget depositedin the soft tissues, particularly in the joints. Such deposits are 5. Xanthine oxidase is an important enzyme known as tophi. This causes that converts hypoxanthine to xanthine, and commonly in the joints resultingin a painful xanthine to uric acid. This enzvme contarns inflammation gouty Sodium urate and/or uric acid FAD, molybdenumand iron, and is exclusively arthritis. may also precipitatein kidneysand uretersthat found in liver and small intestine.Xanthrne resultsin renal damageand stone formation. oxidase liberatesH2O2 which is harmful to the tissues.Catalase cleaves H2O2 to H2O and 02. Historically, gout was found to be often associatedwith high living, over-eating and Uric acid (2,6,8-trioxypurine)is the final alcohol consumption In the previous centuries, excretory product of purine metabolism in alcohol was contaminatedwith lead during its humans. Uric acid can serve as an important manufacture and storage. Lead poisoning leads antioxidant by getting itself converted (non- to kidney damage and decreased uric acid enzymatically)to allantoin.lt is believedthat the excretion causinggout. antioxidantrole of ascorbicacid in primatesis replacedby uric acid, since theseanimals have fhe prevalence of gout is about 3 per 1,000 lost the ability to synthesizeascorbic acid. persons,mostly affecting males. Post-menopausal *harpten'l 7 ; METABOLISMOF NUCLEOTIDES 395 women, however,are as susceptibleas GlYcogen. Grucose men for this disease.Cout is of two types-primary and secondary. 1. Primary gout : lt is an inborn error of metabolism due to overproduction of uric acid. This is mostly relatedto increasedsynthesis of purine nucleotides.The following are the imoortant metabolic defects ()associated with primarygout (Fig.t7.e) . PRPP synthetase : ln normal Ribose5-phosphate circumstances,PRPP synthetaseis I under feedback control by purine I nucleotides (ADP and CDP). I PRPPsvnthetaset However, variant forms of PRPP +I synthetase-whichare not subjected PRPP Glutamine to feedback regulation-have been detected.This leadsto the increased PRPPqlutamvl- productionof purines. amidotrinsferdseI PRPP glutamylamidotransferase : The lack of feedbackcontrol of this 5-Phosphoribosylamine enzyme by purine nucleotidesalso I leadsto their elevatedsynthesis. i HGPRT deficiency : This is an ITPPFJ Hypoxanthine r lno.in" ro*ophosphate enzyme of purine salvagepathway, and its defect causes Lesch-Nyhan "-""'unom | \' "rl syndrome.This disorderis associated Guanine ) GMP AMp<- Adenine with increased synthesis of purine nucleotidesby a two-fold mechanism. Firstly, decreased utilizationof purines(hypoxanthine uypoxlntrine and guanine) by salvage pathway, i I resultingin Xanthineoxidase the accumulationand 7 | diversion of PRPP for purine I nucleotides.Secondlv, the defect in + salvagepathway leadsto decreased lanthine levels of IMP and CMP causing I impairmentin the tightly controlled I \ Xanthineoxidase feedback regulation of their | production. +I Uric acid Glucose 5-phosphatasedeficiency : ln type I glycogen storagedisease Fig. 17.9 : Summary of possible enzyme alterationscausing gout (von Cierke's),glucose 6-phosphate ( I -t ncreased enzyme activily; ; -Decreased enzyme activity ; cannot be converted to glucose GSH-Reduced glutathione; G-S-S-G-Oxidized glutathione; due to the deficiency of glucose PRPP-Phosphoribosyl pyrophosphate; HGPRT-Hypoxanthine- 6-phosphatase.This leads to the gu ani ne phosphoribosyltran sferase). 396 BIOCHEMISTFIY

increasedutilization of glucose 6-phosphate by hexosemonophosphate shunt (HMP shunt), resulting in elevated levels of ribose 5-phosphateand PRPPand, ultimately,purine overproduction.von Gierke's diseaseis also associated with increased activitv of glycolysis.Due to this, lacticacid accumulates in the body which interfereswith the uric acid N excretionthrough renal tubules. N . Elevationof glutathione reductase : Increased H H glutathionereductase generates more NADP+ Allopurinol Alloxanthine which is utilizedby HMP shunt.This causes increased ribose S-phosphate and PRPP Fig. 17.10: Structuresof hypoxanthine and itsstructural analoos. synthesis. Among the five enzymesdescribed, the first three are directly involved in purine synthesis. oxidase.This type of inhibition is referredto as The remainingtwo indirectly regulatepurine suicide inhibition (For more details, Refer production.This is a good exampleto show how Chapter 6). an abnormality in one metabolic pathway Inhibitionof xanthineoxidase by allopurinol influences the other. leadsto the accumulationof hypoxanthineand 2. Secondarygout : Secondaryhyperuricemia xanthine. These two compounds are more is due to various diseasescausing increased soluble than uric acid, hence easily excreted. synthesisor decreasedexcretion of uric acid. Besidesthe drug therapy,restriction in dietary Increaseddegradation of nucleic acids (hence intake of purines and alcohol is advised. more uric acid formation)is observedin various Consumption of plenty of water will also be cancers(leukemias, polycythemia, lymphomas, useful. etc.) psoriasisand increased tissue breakdown (trauma,starvation etc.). The anti-inflammatory drug colchicine is used for the treatmentof gouty arthritis.Other anti- The disordersassociated with impairment in inflammatory drugs-such as phenylbutazone, renal function cause accumulationof uric acid indomethac in, oxyphenbut azone corticostero ids- which may lead to gout. I are also useful. Uric acid pool in gout FseudoEout By administrationof uric acid isotope(N1s), the miscibleuric acid pool can be calculated.lt The clinical manifestationsof pseudogoutare is around 1,200 mg in normalsubjects. Uric acid similarto gout. But this disorderis causedby the pool is tremendouslyincreased to 3,000 mg. or deposition of calcium pyrophosphate crystals in even more/ in patientssuffering from gout. joints. Further/serum uric acid concentrationis normal in pseudogout. Treatment of gout Lesch-Nyhan syndrome The drug of choice for the treatment of primary gout is allopurinol. This is a structural This disorder is due to the deficiency of analog of hypoxanthine that competitively h ypo xa nthi n e-guan i ne ph ospho r ibosy ltran sferase inhibits the enzyme xanthine oxidase. Further, (HCPRT),an enzyme of purine salvagepathway allopurinol is oxidized to alloxanthine by (SeeFig.l7.fl. lt was first describedin 1964 by xanthine oxidase (Fig.l7.l0). Alloxanthine, in Michael Lesch(a medicalstudent) and William turn, is a more effective inhibitor of xanthine L. Nyhan (his teacher).