Intermediary Metabolism

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INTERMEDIARY METABOLISM

Nucleotide metabolism

Prahlad C. Ghosh Professor Department of Biochemistry University of Delhi South Campus Benito Juarez Road New Delhi-110021 E.mail: [email protected]; [email protected]

26-May-2006

CONTENTS What are nucleotides? What are nucleosides? Biosynthesis of purine ribonucleotides Biosynthesis of pyrimidine nucleotides Biosynthesis of deoxyribonucleotides Formation of deoxythymidylic acid from deoxyuridylic acid Mechanism of action of some anticancer drugs Salvage of purine and pyrimidine from catabolic pathways Disease associated with purine metabolism Degradation of purines Degradation of pyrimidines Biosynthesis of nucleotide coenzymes

Significant keywords Nucleotides; Nucleosides; Biosynthesis; Origin of atom; Regulation of purine/pyrimidine biosynthesis; Anticancer drugs; Deoxyribonucleotides; Catabolism; Salvage of purine/pyrimidine; Gout; Lesch Nyhan Syndrome; Allpurinol; Nucleotide coenzyme

What are nucleotides? A nucleotides consists of a heterocyclic nitrogenous base which is a derivatives of either purine or pyrimidine; a pentose sugar, which is either D-ribose or 2-deoxy-D-ribose and one or more phosphate groups. Two purine derivatives (adenine and guanine) and three pyrimidine derivatives (cytosine, uracil and thymine) are the major bases found in nucleotides.

Fig. 1 A: Structure of major purine bases

Fig. 1B:Structure of major pyrimidine bases

In addition to these major bases, a number of other purine and pyrimidine derivatives have been isolated in small amount from various nucleic acids. The transfer RNAs (t-RNAs) contain several unusual bases, such as methylated or dimethylated derivatives of adenine, guanine, cytosine and uracil. Other important purine occurs in living organisms; these include hypoxanthine, xanthine and uric acid, which are catabolic products of adenine and guanine metabolism.

What are nucleosides? A nucleoside consists of a purine or pyrimidine base linked to a D-ribose or 2-deoxy-D- ribose via a N-β -glycosidic bond. The glycosidic C-1 carbon atom of the pentose is linked to the nitrogen in position 9 of the purine or to the nitrogen in position 1 of the pyrimidine. The predominant ribonucleosides are adenosine, guanosine, cytidine and uridine; the predominant deoxyribonucleosides are deoxyadenosine, deoxyguanosine and deoxycytidine and deoxythymidine.

Figure 2A: Structure of ribonucleosides

Figure 2B: Structure of Deoxyribonucleosides

The nucleotides are sugar-O-phosphate esters of nucleosides, the most common site of esterification being the 5-hydroxyl group of the sugar. Such compounds are called ribonucleside-5'-monophosphates and dedoxyribonucleoside -5'-monophosphate. The living organisms do not require purines and pyrimidines in the diet but can synthesize them de novo from the products of protein and carbohydrates metabolism.

Fig. 3: Structure of ribonucleotides and deoxyribonucleotides

Biosynthesis of purine ribonucleotides In 1943 Schoenheimer et al gave ammonia labeled with N15 to rats and pigeons and found the N15 to be rapidly incorporated into the purines and pyrimidines of nucleic acids of the internal organs. These experiments indicate that purines and pyrimidines are synthesized from simple molecules. What are the simple molecules required for purine and pyrimidine nucleotide biosynthesis?

Origin of atoms in purine ring The biosynthetic origin of the purine base came form the experiments of J.M. Buchanan and his Colleagues. They fed various possible isotopic precursors to pigeons and determined the sites of incorporation of the labeled atoms into the purine ring. Pigeons were chosen for such experiments since they excrete uric acid, a purine derivative that is easily isolated in

4 pure form, labeled in definite positions. Chemical degradation of the uric acid revealed the origin of the atoms in the purine ring.

Fig. 4: Origin of atoms in purine ring.

Nitrogen atoms 3 and 9 arise from the amide group of glutamine, nitrogen 1 from aspartate and nitrogen 7 from glycine. Carbon atoms 4 and 5 also derive from glycine, indicating that the backbone of the glycine molecule is directly incorporated into the purine ring. Carbon atoms 2 and 8 are furnished by formate and carbon 6 by CO2. However, several years of research were required to elucidate the complex enzymatic steps involved in the biosynthesis of the purine nucleotides.

How and which sequences purines are synthesized? There are several derivatives of purine present in the body, which one is synthesized first? To answer these questions, Mike Buchanan and Greenberg (1945-50) performed a kinetic study of incorporation of 14C-formate into purine ring at different time intervals. They fed 14C-formate to rats and after different time intervals rats were sacrificed, liver tissues were exercised and analyzed for purine nucleotides in the liver. They observed that radiolabeled inosinic acid (IMP) appeared first in the tissue followed by both adenylic acid (AMP) and guanylic acid (GMP) in equal amount. Other nucleotides arise latter on and free bases arise much latter on. So it was suspected that IMP is the first product in the pathway of purine biosynthesis.

From these studies it was concluded that purines are synthesized de novo not as free purines but first as the nucleotide inosinic acid (hypoxanthine-ribose-5'-phosphate), which is then converted into the adenine and guanine nucleotides.

Enzymatic steps involved in purine nucleotide biosynthesis Starting precursor It was found that the starting material for inosinic acid biosynthesis is an activated form of α-D-ribose-5'-phosphate, on which a purine ring is built up step by step, resulting directly in the formation of a nucleotide.

Activation of α-D-Ribose-5-Phsophate The biosynthetic pathway to adenylic and guanylic acids begins with the activation of α-D- ribose-5'-phosphate by enzymatic pyrophosphorylation at the expense of ATP to form 5'- phospho-α-D-ribose-1-pyrophosphate (PRPP) This is an unusual reaction in that the pyrophospahte group of ATP is transferred intact.

Fig. 5: Activation of α–D-ribose-5'-phosphate to PRPP

The energy-rich metabolite phosphoribosyl pyrophosphate (PRPP) is then converted to inosine monophosphate (IMP), the precursor of AMP and GMP, by a ten steps pathway.

Pathway to IMP from PRPP 1. Glutamine-PRPP amidotransferase catalyzes displacement of the 1-pyrophosphoryl group of PRPP by NH2 derived from the amide group of glutamine to yield 5'- phospho-β -D-ribosyl -1-amine. The amide nitrogen atom of the glutamine is the first atom of the purine ring to be introduced; it correspond to nitrogen atom 9 of the finished purine ring. In this step, the anomeric carbon atom 1 of the D-ribose undergoes inversion from the α to the β configuration. The β- configuration so introduced is retained in the final purine product. 2. C-4, C-5 and N-7 of the imidazole portion of the purine are derived from glycine through the action of phosphoribosylglycinamide synthetase. This enzyme catalyzes the formation of an amide bond between the C-1 amino group of 5'- phosphoribosylamine and the carboxyl group of glycine to form 5'- phosphoribosylglycinamide. ATP is hydrolyzed to ADP and Pi during the course of reaction. After only two reactions in the sequence, four of the nine atoms of the purine are introduced; the remaining atoms are contributed one at a time. 3. The free amino group of 5'-phosphoribosylglycinamide reacts with the formyl carbon of N10-formyltetrahydrofolate to form 5'-phosphoribosyl N- formylglycinamide. This reaction is catalyzed by phosphoribosyglycinamide formyltransferase and releases tetrahydrofolate. The formyl carbon becomes C-8 of the purine. 4. In the next step in purine nucleotide biosynthesis involves the introdiuction of nitrogen atom 3, which is derived from the amide group of glutamine, resulting in the formation of 5'-phosphoribosyl-N-formyl-glycinamide. The transfer of the amino group from glutamine requires input of energy from ATP, which is cleaved to ADP and Pi. 5. Phosphoribosyl-aminoimidazole synthetase catalyzes closure of the imidazole ring by elimination of H2O to yield 5'-phosphoribosyl-5 aminoimidazole. This reaction also requires input of energy from ATP.

6 6. Carbon atom 6, which arises from CO2, is now introduced in a carboxylation reaction catalyzed by 5'-phosphoribosyl-5-aminoimidazole carboxylase. This reaction is somewhat unusual since neither biotin nor ATP is required for carboxylation. 7. Phosphoribosyl-aminoimodazole–succinocarboxamide synthetase catalyzes formation of an amide link between the carboxyl group of 5'-phosphoribosyl-4 aminoimidazole 4-carboxylate and aspartate with the formation of 5'- phosphoribosyl-4- (N-succinocarboxamide)-5-aminoimodazole. The nitrogen atom introduced becomes nitrogen 1 of the purine ring. 8. Adenylosuccinate lyase catalyzes elimination of fumarate from the above product to yield 5'-phosphoribosyl 4—carboxamide 5-aminoimidazole. 9. The remaining carbon atom of the purine ring is now introduced by transfer of the formyl group of N10-formyl-FH4. This reaction is catalyzed by phosphoribosyl- aminoimidazole-carboxamide formyltranferase and the product formed is 5'- phosphoribosyl-4-carboxamide-5-formamidoimidazole. 10. The pyrimidine portion of the purine ring system is then closed by elimination of H2O to form the ribonucleotide inosinic acid (IMP), the first product in this biosynthetic pathway to posses a complete purine ring system. This reaction is catalyzed by IMP cyclohydrolase.

Fig. 6: Reactions of the de novo pathway for the biosynthesis of inosine-5'- monophosphate

Six high-energy phosphate group of ATP are utilized fro the formation of inosinic acid from D-ribose-5'-phosphate if it is assumed that the PPi group displaced from PRPP is ultimately hydrolyzed to Pi by pyrophosphatase. The purine base part of inosinate is called hypoxanthine.

Conversion of IMP to AMP and GMP. IMP, synthesized de novo by the 10-step pathway, is a branch-point metabolite that can be converted either to AMP or GMP. The synthesis of AMP from IMP requires only the carbonyl group at C-6 to an amino group. Two steps are required for this transformation. First, adenylosuccinate synthetase catalyzes the GTP-dependent condensation of aspartate and IMP to yield adenylosuccinate. Then AMP is produced and fumarate is eliminated in a reaction catalyzed by adenylosuccinate lyase.

The conversion of IMP to GMP also requires two steps. First, NAD-dependent IMP- dehydrogenase catalyzes the addition of a keto group to C-2 of IMP to yield xanthylic acid. Next, GMP synthetase catalyzes conversion of the keto group to an amino group in a

8 reaction that requires glutamine as amino donor, producing GMP. ATP is converted to AMP and pyrophosphate during the reaction.

(A) (B) Fig. 7: The pathway from inosinic acid to adenylic acid (A) and guanylic acid (B)

Conversion of GMP and AMP to GTP and ATP The adenylic and guanylic acids are converted into GTP and ATP by two enzymatic reactions catalyzed by nucleoside monophosphate kinase and nucleoside diphosphate kinase, respectively. GMP + ATP = GDP + ADP ADP + ATP = GTP + ADP

Regulation of purine nucleotide biosynthesis There are two levels of regulatory control over purine nucleotide biosynthesis: 1. The first involves regulation of the pathway leading to biosynthesis of inosinic acid (IMP) and hence provides common control over the biosynthesis of all purine nucleotides. 2. The second involves regulation of the branch pathways from IMP to adenylic and guanylic acids and serves to regulate the relative abundance of AMP and GMP.

Fig. 8: Regulation of purine nucleotide biosynthesis

Regulation of inosinic acid biosynthesis is exerted at the early two reactions steps leading to the formation of PRPP and 5'-phosphoribosylamine. The enzyme PRPP kinase that catalyzes the synthesis of PRPP from ribose-5'-phosphate is inhibited by IMP, AMP and GMP. Inhibition of the synthesis of PRPP is an important regulatory mechanism because PRPP concentrations influence the rate of biosynthesis of purine nucleotides. The enzyme glutamine-PRPP amidotransferase, which catalyzes transfer of an amino group to PRPP, is a bivalent regulatory enzyme, is inhibited by ATP, ADP and AMP or by GTP, GDP and GMP, each of the two types of nucleotides apparently binding to a separate allosteric site on the enzyme. This is an example of cumulative-feedback inhibition because the net inhibition

10 caused by these nucleotides together is greater than the sum of the individual nucleotides acting alone.

Regulation of the pathway from IMP to AMP and from IMP to GMP is achieved in two ways: 1. Hurry-Up Process: The reaction pathway leading from xanthylic acid to guanylic acid (GMP) requires ATP as a reactant, whereas the pathway leading from inosinic acid to adenylic acid (AMP) requires GTP. Thus an excess of ATP accelerates the pathway leading to guanylic acid; similarly, an excess of GTP accelerates the synthesis of adenylic acid. This reciprocal arrangement helps to balance the rates of production of AMP and GMP. 2. Slow-Down Process: A second type of coordination between AMP and GMP levels is provided by two feedback inhibition. Two enzymes of the pathway leading to AMP from IMP and GMP from IMP are allosteric in nature. AMP is an allosteric inhibitor of adenylosuccinate synthetase, which catalyzes the conversion of IMP into adenylosuccinic acid,, GMP is an allosteric inhibitor of IMP dehydrogenase, which catalyzes the conversion of IMP into xanthylic acid. In this process AMP and GMP retard their own synthesis.

Biosynthesis of pyrimidine nucleotides Origin of atoms of pyrimidine ring The sources of the atoms of pyrimidine synthesized by de novo pathway are shown in figure. C-2 and N-3 of the pyrimidine are provided by glutamine and bicarbonate, respectively. The remaining atoms, N-1, C-6, C-5 and C-4 are contributed by aspartate.

C4 Glutamine N3 5C Aspartic acid CO2 C2 6C N1

Fig. 9: Origin of atoms of pyrimidine ring

Difference between biosynthesis of purine and pyrimidine nucleotides The biosynthetic pathway leading to the pyrimidine nucleotide is much simpler than the pathway to the purine nucleotides. It differs from the purine pathway in that the de novo pathway of pyrimidine begins with construction of the pyrimidine ring first and the D- ribose-5-phospahte is attached with the base at the end.

Starting precursor and activation of precursors

The starting precursors for pyrimidine ring are glutamine and CO2. The synthesis of pyrimidine ring starts with an ATP-dependent reaction between glutamine and CO2 catalyzed by carbamoyl phosphate synthetase with formation of a very unstable activated compound carbamoyl phosphate. C-2 and N-3 of the pyrimidine are provided by glutamine

11 and bicarbonate, respectively. The carbamoyl phosphate is also an intermediate in the synthesis of urea. The synthesis of this activated compound is compartmentalized in eucaryotes. Carbamoyl phosphate consumed in the synthesis of pyrimidines is formed in the cytosol, whereas that used in the synthesis of urea is formed in mitochondria. There are two distinct carbamoyl phosphate synthetase.

2ATP + Glutamine + CO2 + H2O Carbamoyl phosphate synthetase

H2N─ C─ O─ P – OH + ADP + Pi

O O (Carbamoyl-phosphate)

Fig. 10: Biosynthesis of carbamoyl phosphate

Another noteworthy difference is that glutamine rather than NH3 is the nitrogen donor in the cytosol synthesis of carbamoyl-phosphate.

Pathway of pyrimidine ring formation from carbamoyl-phosphate 1. The pyrimidine ring is formed by the condensation of aspartate with carbamoyl- phosphate that produces N-carbamoyl-L-aspartate. This reaction is catalyzed by aspartate transcarbamoylas (ATCase) and contributes N-1, C-6, C-5 and C-4, which are the remaining atoms of pyrimidine ring. 2. In the second step of pyrimidine biosynthesis the ring is closed by removal of H2O from N-carbamoyl- L-aspartate by the action of Zn+-containing enzyme dihydroorotase to yield L-dihydroorotic acid. 3. L-Dihydroorotic acid is oxidized to orotic acid by the flavoprotein NAD-dependent orotate dehydrogenase. 4. The next step in the biosynthesis of pyrimidine nucleotides is the acquisition of a ribose-phosphate group. Orotate reacts with PRPP to form orotidine 5'- monophosphate. This reaction is catalyzed by orotate phosphoribosyltransferase and driven forward by the hydrolysis of pyrophosphate release in this reaction. 5. In the final step of UMP biosynthesis is a decarboxylation reaction, catalyzed by orotidine-5'- monophosphate decarboxylase, which produces uridine 5'- monophosphate (UMP). UMP can serve as a precursor for all the other pyrimidine nucleotides, including those containing cytosine or thymine.

Biosynthesis of CTP from UTP UMP is the precursor of the cytidine nucleotides. Three reactions are required for the conversion of UMP to CTP. First, nucleoside monophosphate kinase catalyzes phosphoryl- group transfer from ATP to UMP, generating UDP and ADP. Then, nucleoside diphosphate kinase catalyzes phosphoryl-group transfer from ATP to UDP, producing UTP and ADP. UMP + ATP = UDP + ADP UDP + ATP = UTP + ADP

12 Nucleoside diphosphate and triphosphate are interconverted by nucleoside diphosphate kinase, an enzyme that has broad specificity.

UTP then undergoes amination by ammonia (bacteria) or glutamine (mammals) at the 4 position of the pyrimidine ring to yield cytidine 5-triphosphate or CTP. This reaction is catalyzed by CTP synthetase and ATP is cleaved to ADP and Pi.

Fig. 11: Biosynthesis of uridylic acid

Fig. 12: Amination of UTP by CTP synthetase

Regulation of pyrimidine nucleotide biosynthesis Although the six catalytic steps leading to the formation of UMP are quite similar in all organisms, the structural organization and regulation of the enzyme involved vary widely among organisms. There is a significant variation in the regulation of pyrimidine nucleotide biosynthesis in E.coli and mammals. In both E.coli and mammals, the enzyme that catalyzes the first committed step in the de novo pathway for the biosynthesis of pyrimidine nucleotide is the target for allosteric regulation. In E.coli, ATCase catalyzes the first committed step leading to UMP, which is the primary regulatory enzyme for the pathway. In mammals, carbamoyl phosphate synthetase II catalyzes the first committed step in the biosynthesis of UMP and is the key regulatory enzyme.

In both E.coli and mammals, the final products of the synthesis of pyrimidine nucleotides, CTP and UTP, exert negative-feedback inhibition: CTP is the principal inhibitor in E.coli and whereas UTP is the principal inhibitor in mammals. ATP acts as an activator for ATCase in E.coli, high level of ATP displaces CTP from the enzyme so that it cannot exert its inhibitory effect and UMP acts as an inhibitor of carbamoyl-phosphate synthetase. In mammals, PRPP, a substrate for the last step of the pathway, acts as an activator for the reaction catalyzed by carbamoyl-phosphate synthetase II.

Fig. 13: Regulation of pyrimidine nucleotide biosynthesis

Biosynthesis of deoxyribonucleotides The 2'-deoxyribonucleotides dATP, dGTP, dCTP and dTTP are the building blocks for DNA and are synthesized through the reduction of ribonucleotides. The 2'-hydroxyl group of ribonucleotides is replaced by a hydrogen atom to provide the 2'-deoxyribonucleotides from which DNA is synthesized. The enzyme catalyzed this reaction is ribonucleotide reductase. The substrate is ribonucleotide diphosphate or triphosphate, and the ultimate reductant is NADPH. The same active site acts on all four ribonucleotides. The overall reaction is

Ribonucleotide diphosphate + NADPH + H Deoxyribonucleotide diphosphate + NADP + H2O

The conversion of these ribonucleotide diphosphates to their deoxy counter part is the first committed step in the biosynthesis of DNA. The actual reaction mechanism is more complex than implied by the equation. It was shown that in E.coli the electron from NADPH are transferred to the substrate through a series of carrier.

NADPH first transfers electrons to FAD, a cofactor bound to thioredoxin reductase. In turn, a disulfide on the enzyme is reduced, and the reduced thioredoxin reductase then passes these electrons to thioredoxin, reducing the disulfide group of thioredoxin to two sulfhydryl groups. Reduced thioredoxin, a 12 Kd protein, in turn reduces the disulfide group of inactivated ribonucleotide reductase to two sulfhydryl groups, thereby activating the enzyme. Activated ribonucleotide reductase then transfers electrons to ribonucleside diphosphate, reducing the 2-hydroxyl group of the ribonucleotide to a hydrogen atom to form the deoxyribonucleotide. The three deoxyribonucleotides dADP, dGDP and dCDP are converted to dATP, dGTP and dCTP by nucleoside diphosphate kinase.

Fig. 14: Summary of the steps in the formation of deoxyribonucleotides

It was thought for some time that thioredoxin is the only carrier of reducing power to ribonucleotide reductase. However, a mutant of E coli totally devoid of thioredoxin is able to reduce ribonucleotide diphosphates to deoxyribonucleotides. This surprising observation led to the discovery of a second carrier system. This alternative system is similar to the system describes above, except that thioredoxin reductase and thioredoxin are replaced by glutaredoxin reductase and glutaredoxin, respectively.

Fig. 15: Summary of the steps in the formation of deoxyribonucleotides in E.coli that lacks thioredoxin reductase

A second type of ribonucleotide reductase is found in certain species of Lactobacillus, Rhizobium, Euglena and Clostridium. This enzyme preferentially uses ribonucleside triphosphates as substrate rather than diphosphate. In this type of ribonucleotide reductase, there is only one type of subunit, and the enzyme has neither Fe3+ nor a tyrosine radical. Moreover it require coenzyme form of vitamin B12 and can use either thioredoxin or dihydrolipoic acid as reducing agent.

Formation of deoxythymidylic acid from deoxyuridylic acid Deoxythymidylate (dTMP), rather than deoxyuridylate (dUMP), is a component of DNA. The only difference between uracil and thymine is the presence of a methyl group in position 5 of pyrimidine ring. The conversion of the uracil residue of dUMP to thymine to form dTMP is essential to the biosynthesis of DNA. This conversion is carried out by

16 thymidylate synthetase in a reaction that requires N5, N10-methylenetetrahydrofolate. The methyl group donor in this reaction is N5, N10-methylenetetrahydrofolate rather than S- adenosylmethionine. The methyl group inserted into deoxyuridylate is more reduced than the methylene group present in tetrahydrofolate derivatives. What is the source of electrons for this reduction? The source of two electrons for the reduction of the methylene group to a methyl group is a hydride ion (H-) from the tetrahydropyrazine ring of N5, N10- methylenetetrahydrofolate itself. N5, N10-Methylenetetrahydrofolate is therefore, both a one carbon donor and an electron donor in the reaction. During the course of the reaction, N5, N10-methylenetetrahydrofolate is oxidized to 7,8-dihydrofolate, which is converted back to N5,N10-methylenetetrsahydrofolate in two steps. First, NADPH-dependent dihydrofolate reductase oxidizes 7,8-dihydrofolate to tetrahydrofolate; then, serine hydroxymethyltransferase contributes a carbon from serine to resynthesize N5, N10- methylenetetrahydrofolate from tetrahydrofolate.

Fig. 16: Formation of deoxythymidylic acid from deoxyuridylic acid

Mechanism of action of some anticancer drugs Rapidly dividing cells require an abundant supply of dTMP to carry our DNA synthesize. They are therefore highly susceptible to agents that inhibit the formation of dTMP. Two enzymes, thymidylate synthetase and dihydrofolate reductase which are directly involved in the biosynthesis of dTMP, are choice target enzymes. Inhibitors of these enzymes are well known anticancer drugs. For example, 5-fluorouracil (fluorodeoxyuridine), a clinically useful anticancer drug, is converted to 5-fluorodeoxyuridylic acid by a pyrimidine salvage pathway. 5-Fluorouridylic acid resembles a substrate for thymidylate synthetase, but the final step of the reaction, elimination cannot occur; the enzyme is irreversibly covalently bound to the fluoro analog and is thereby totally inhibited. The division of cancer cells is slowed when 5-fluorouracil is administered because the cancer cells cannot produce the dTMP required for DNA synthesis. This is an example of a suitable inhibitor, in which an

17 enzyme converts a substrate into a reactive inhibitor that immediately inactivates its catalytic activity.

The synthesis of dTMP can also be blocked by inhibiting the regeneration of tetrahydrofolate. This can be achieved by treating the cells by analogs of dihydrofolate, such as aminopterin and amethopterin (methotrexate). These dihydrofolate analogs are potent competitive inhibitors of dihydrofolate reductase that reduces dihydrofolate to tetrahydrofolate. These two analogs of folate bind tightly to dihydrofolate reductase, preventing the formation of the N5, N10-methylenetetrahydrofolate needed for conversion of dUMP to dTMP. Consequently, cancer cells perish by a process termed thymineless death. Normal cells can be affected also, but they are usually are growing more slowly and so are less sensitive to the antifolate drugs.

Fig. 17: Mechanism of action of some anticancer drugs

Regulation of deoxyribonucleotide biosynthesis The reduction of ribonucleotide diphosphate is precisely controlled by allosteric interactions. The ribonucleotide reductase in E coli is the most common type of ribonucleotide reductase. It is composed of two nonidentical subunits called B1 (mol wt 160Kd dimer) and B2 (mol.wt 78 Kd dimer), which together form the active site of the enzyme. Each subunit of B1 protein contains two types of regulatory sites- a substrate specificity site and an activity site. Deoxyribonucleoside triphosphates, which are allosteric effectors of the enzyme, bind to these regulatory sites. The sulfhydryl groups that serve as electron donor to reduce the 2'-hydroxyl group of the ribonucleside diphosphate substrates also are located on protein B1. Each subunit of B2 protein contains an active site tyrosine residue. The p-hydroxyphenyl group of one of the active-site tyrosine residues is a stable free radical, which is directly involved in the reduction of the 2'-hydroxyl group. This free radical is stabilized by an iron center composed of two Fe3+ atoms coordinated by an oxygen atom.

18 In E coli, the activity of ribonucleotide reductase is regulated allosterically by several nucleotides, which bind to the regulatory sites. Binding of ATP to an activity site activates the catalytic site of the enzyme for the reduction of CDP and UDP; binding of dATP to the activity site inactivates the enzyme for all substrates. Binding of dTTP to the substrate site inhibits the reduction of CDP and UDP, whereas it activates the reduction of GDP and ADP. Binding of dGTP to the substrate specificity site also stimulates reduction of GDP and ADP. The result of this regulation is a balanced production of the 2'- deoxyribonucleotides required for DNA synthesis.

Fig. 18: Model of Ribonucleotide reductase

Fig. 19. Regulation of deoxyribonucleotide biosynthesis

Salvage of purine and pyrimidine from catabolic pathways Nucleotides of a cell undergo continuous turnover. Nucleotides are hydrolytically degraded to nucleosides by nucleotidases. Phosphorolytic cleavage of nucleosides to free bases and

19 ribose 1-phospahte (or deoxyribose 1-phosphate) is catalyzed by nucleoside phosphorylase. Ribose 1-phosphate is isomerized by phosphoribomutase to ribose 5-phospahte, a substrate in the synthesis of PRPP.

Salvage of purines Free purines formed from nucleotides are salvaged in vertebrates for reuse in nucleotide and nucleic acid biosynthesis. The salvage pathway is much simpler and less costly than the reactions of de novo pathways. In the salvage reaction, the ribose phosphate moiety of PRPP is transferred to the purine to form the corresponding nucleotides.

There are two well known salvage enzymes with different specificities. Adenine phosphoribosyltransferase catalyzes synthesis of AMP from adenine and PRPP, and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyzes the synthesis of IMP and GMP from their respective free base and PRPP.

Adenine + PRPP AMP + PPi Hypoxanthine/Guanine + PRPP IMP/GMP

Since up to 90% of the free purines formed by man are salvaged and recycled, these salvaged pathways are very important in the purine economy of vertebrates.

Salvage of pyrimidines As in the biosynthesis of purine nucleotides, the salvage of pyrimidines is far more energy efficient than de novo biosynthesis. In microorganisms, pyrimidine salvage pathways reuse pyrimidines to form nucleotides. For example, in certain bacteria UMP is synthesized from uracil by phosphoribosyltransferase in presence of PRPP.

Uracil + PRPP UMP + PPi

Mammals, however, do not salvage pyrimidine base in significant amounts by this pathway. An alternative route for conversion of uracil to UMP, both in bacteria and in higher animals, is by successive reactions catalyzed by uridine phosphorylase and uridine kinase, respectively

Uracil + ribose 1-phosphate → uridine + Pi Uridine + ATP → UMP +ADP

Cytidine is also act as substrate for uridine kinase Cytidine + ATP → CMP + ADP

Thymidine is similarly converted to dTMP according to the following reactions: Thymine + deoxyribose 1-phosphate → thymidine + Pi Thymidine + ATP → dTMP + ADP

A deoxycytidine kinase catalyzes the following reaction: Deoxycytidine + ATP → dCMP + ADP

20 Deoxyadenosine and deoxyguanosine are also substrate for deoxycytidine kinase, but the purine deocyribonucleosides have much higher Km values than that of deoxycytidine.

Disease associated with purine metabolism Two well-known diseases are associated with metabolism of purines: Lesch Nyhan Syndrome and Gout.

Gout: Gout is a disease that affects joints and leads to arthritis. The major biochemical feature of gout is an elevated level uric acid in the serum. Inflammation of the joints is trigger by the precipitation of sodium urate crystals. Kidney disease may also occur because of the deposition of urate crystal in that organ. Gout primarily affects adult males. The biochemical lesion in most cases of gout has not been elucidated. It seems likely that gout is an expression of a variety of inborn error of metabolism in which excessive production of urate is common finding. Many patients have a partial deficiency of HGPRT, the enzyme that catalyzes the salvage of hypoxanthine and guanine. A deficiency of this leads to reduced synthesis of GMP and IMP by the salvage pathway. The consequent increase in the level of PRPP markedly accelerate purine biosynthesis by de novo pathway. The formation of 5-phosphoribosyl-1-amine, the first committed intermediate, is normally limited by the availability of PRPP. Excessive PRPP also interferes with feedback inhibition of the amidotransferase that catalyzes this step. A few patients with gout have an abnormally high level of PRPP kinase. The allosteric control of this enzyme is impaired in these patients. This results in excessive production of PRPP, which in turn accelerates the rate of de novo synthesis of purine.

Treatment of Gout: To treat gout, allopurinol is used to decrease the production of uric acid through the inhibition of xanthine oxidase. Allopurinol is an analog of hypoxanthine in which the position of N-7 and C-8 are interchanged. The mechanism of action of allopurinol is very interesting: it acts first as a substrate and then as an inhibitor of xanthine oxidase. The enzyme hydroxylates allopurinol to alloxanthine (oxipurinol), which then remains tightly bound to the active site by forming a chelate with a Mo4+ of xanthine oxidase. The chelate of xanthine oxidase with oxipurinol traps the enzyme in its reduced state and renders it inactive. Thus, uric acid cannot be formed from hypoxanthine and xanthine and these bases are excreted instead, and the symptoms of gout can be relieved. This mode of action of allopurinol is an example of suicide inhibitor. There is significant decrease in the total rate of de novo purine biosynthesis in the patients following administration of allopurinol. This inhibitory action of allopurinol depends on its interaction with PRPP to form allopurinol-ribonucleotide. Consequently, the level of PRPP, the limiting substrate in the de novo biosynthesis of purine, is lowered. Furthermore, high level of allopurinol- ribonucleotide inhibits the conversion of PRPP into phosphoribosylamine by phosphoribosyl-amidotransferase.

Lesch Nyhan Syndrome: This is an inherited neurological disease of children that is characterized by mental retardation and self-mutilation. The most striking expression of this inborn error of metabolism is compulsive self-destructive behavior. At age two or three, children with this disease begin to bite their fingers and lips. The tendency to self –mutilate is so extreme that it is necessary to protect these children by such measures as wrapping their hands in gauge. Those afflicted also tend to be aggressive towards others. The clinical conditions result from the complete absence of one salvage pathway enzyme hypoxanthine- guanine phosphoribosyltransferase (HGPRT). When this enzyme is deficient, hypoxanthine

21 and guanine are not salvaged to their respective nucleotides, IMP and GMP and hence are degraded to uric acid. The rate of de novo biosynthesis of purine nucleotides is abnormally high; therefore catabolism of purines is further increased, and uric acid is overproduced. In adult life, persons afflicted with this disease suffer from severe gout. It is not known how the severe neurological disturbance of Lesch Nyhan Syndrome is related to a relatively simple defect of the purine salvage pathway. However, the level of HGPRT is abnormally higher in the brain than in any other tissue. In contrast, the activity of the amidotransferase that catalyzes the committed step in the de novo pathway is rather low in the brain. The brain may be very dependent on the salvage pathway for the synthesis of IMP and GMP. This may be one of the several reasons for mental retardation of the patient.

Fig. 20: Structure of allopurinol and hypoxanthine

Allopurinol is highly effective in diminishing urate synthesis in the Lesch Nyhan Syndrome patients. However, patients with this disease do not convert allopurinol into the ribonucleotide because they lack HGPRT. Hence, the administration of allopurinol does not lower their level of PRPP and so de novo purine synthesis is not diminished. Furthermore, allopurinol fails to alleviate the neurological symptoms.

Another disease associated with purine metabolism is severe immunodeficiency disease in which T-lymphocytes and B-lymphocytes do not develop properly. This disease is caused due to the deficiency of adenosine deaminase. Absence of adenosine deaminase leads to a 100-fold increase in the cellular concentration of dATP, which is strong inhibitor of ribonucleotide reductase. High level of dATP produces a general deficiency of other

22 deoxyribonucleotides in T-lymphocytes and B-lymphocytes. The basis for lymphocytes toxicity is less clear. These patients lack an effective immune system and subsequent inability to combat infections and do not survive unless isolated in a sterile environment.

Degradation of purines In higher animals nucleotides resulting from degradation of the nucleic acids by the action of nucleases usually undergo enzymatic hydrolysis to yield the free purine and pyrimidine bases. If not salvaged and reused, the free bases are degraded further and the end products excreted. In the first step in the degradation of purine nucleotides, nucleotides are converted to nucleosides by the action of nucleotidases. The nucleosides so formed then converted to free bases and ribose- or deoxyribose1-phosphate by the action of nucleoside phosphorylase. The major purines adenine and guanine are first converted into xanthine, which is then converted to uric acid.

Fig. 21: The pathway for the degradation of AMP, GMP and IMP to uric acid

A very active guanine deaminase converts guanine to xanthine. In case of adenine, an active deaminase does not seem to exist in mammals and deamination occurs at the levels of AMP and adenosine by the action of AMP deaminase and adenosine deaminase and ultimately converted to hypoxanthine by the action nucleotidases and purine nucleoside phosphorylase. Hypoxanthine and xanthine are then converted to uric acid by the action of the iron and molybdenum containing flavin enzyme, xanthine oxidase. Molecular oxygen, the oxidant in the reaction is reduced to H2O2, which is decomposed to H2O and O2 by catalase. In human, urate is the final product of purine degradation and is excreted in the urine.

Mammals other than primates oxidize uric acid further to allantoin. Human and other primates as well as birds lack urate oxidase and hence excrete uric acid as the final product of purine catabolism. In many animals other than mammals, allantoin is metabolized further to other products that are excreted, for example, allantoic acid (some teleost fish), urea (most fishes, amphibians, some mollusks), and ammonia (some marine vertebrates).

Fig. 22: Degradation of uric acid to excretory products

Degradation of pyrimidines The pathways of degradation of CMP and UMP to uracil and conversion of uracil to β– alanine are shown in figure 23. All three pyrimidine nucleotides are converted to their respective nucleosides – cytidine, uridine and deoxythymidine by a general 5'-nucleotidase reaction as described earlier. Cytidine or 2'-deoxycytidine is then loses its amino group to form uridine or deoxyuridine in a reaction catalyzed by cytidine deaminase. In some microorganism, this removal of amino group occurs at the level of cytosine, as it is converted to uracil. Uridine and deoxyuridine are converted to their respective bases by a phosphorylase reaction, which salvages the pentose in the form of ribose-1-phosphate or deoxyribose-1-phosphate.

Fig. 23A: Degradation of cytosine

Fig. 23B: Degradation of thymine

Enzymes present in mammalian liver are capable of the catabolism of both uracil and thymine. Both uracil and thymine are reduced in NADPH-dependent reactions catalyzed by dihydrouracil dehydrpgenase to produce dihydrouracil and dihydrothymine, respectively. Hydrolysis between N-3 and C-4 of the heterocyclic ring is catalyzed by dihydropyrimidinase, which converts dihydrouracil to N-carbamoyl-β-alanine and dihydrothymine to N-carbamoyl -β-isobutyrate. Finally the carbamoyl group is hydrolyzed off from the product to yield β-alanine and β-aminoisobutyrate, respective, from uracil and thymine by the action of an amidohydrolase. These products can be converted to acetyl-

26 CoA and succinyl-CoA, respectively. Alternatively β-alanine is used in the synthesis of pantothenate, which then is used in the synthesis of coenzyme A.

Biosynthesis of nucleotide coenzymes Some enzymes depend for activity only on their structure as protein, while others also require one or more nonproteinous organic components, called coenzymes. The coenzymes play an important role in metabolism, which we have already discussed. Nucleotide coenzymes are coenzymes, which have nucleotide as nonproteinous components, such as flavin nucleotides, NAD+, NADP+, and coenzyme A. We will discuss the pathway for the biosynthesis of each coenzyme.

Biosynthesis of NAD+ and NADP+ The nicotinamide moiety of the coenzymes nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) synthesize by several routes. In liver and other tissues, tryptophan degradation forms, among many products, quinolinic acid, which is converted to nicotinate mononucleotide (deamidonicotinamide mononucleotide, deamido-NMN) by quinolinate phosphoribosyltransferase (Fig. 24). In the cytosol of many cells of many tissues, and in yeast and other organisms, there is present a nicotinate phosphoribosyltransferase that also forms deamido-NMN. A very similar phosphoribosyltransferase present in the cytosol of all animal tissues investigated acts on nicotinamide (Fig. 24). These transferases are responsible for utilization of nicotinate and nicotinamide in the diet. The role of ATP in these reactions is unclear. Some transferases do not require it, for other it seems to be an allosteric regulator, and in yet other cases ATP seems to be hydrolyzed to yield ADP and Pi in equimolar amounts with deamido-NMN formation.

The mononucleotides so formed are converted to the corresponding dinucleotides by NMN- adenyltransferase. In mammalian cells it appears to be a single enzyme that catalyzes both reactions, but adenyltransferase acting only on NMN has been isolated from some bacteria. A cytoplasmic NAD+ synthase present in yeast, and tissues transfer the amino group from glutamate at the expenses of ATP hydrolysis to form NAD+ (Fig. 24).

NADP+ is derived from NAD+ by phosphorylation of the 2-hydroxyl group of the adenine ribose moiety. This transfer of a phosphoryl group is catalyzed by NAD+-kinase. NADH is not a substrate for this enzyme and inhibits competitively with respect to NAD+.

NAD+-kinase NAD+ + ATP NADP+ + ADP

Human can synthesize the required amount nicotinate if the supply of tryptophan in the diet is adequate. However, an exogenous supply of nicotinate is required if the dietary intake of tryptophan is low. Pellagra is a deficiency disease caused by a dietary insufficiency of tryptophan and nicotinate.

Fig. 24: Biosynthesis of NAD+

Biosynthesis of Flavin Mononucleotide (FMN) and Dinucleotide (FAD) Riboflavin (Vit.B2) is synthesized by microorganisms fungus, yeast. However it is an essential dietary constituent for mammals and is converted in the body to the mononucleotide and dinucleotide forms that function as the prosthetic groups of many enzymes. Riboflavin is converted to riboflavin-5-phosphate, more commonly called flavin mononucleotide (FMN), by flavokinase as shown in Fig. 25. This enzyme is present in yeast, plants and variety of animal tissues.

The other nucleotide form of riboflavin, flavin adenine dinucleotide (FAD) is formed from FMN in a reaction catalyzed by FMN-adenyltransferase. This enzyme is widely distributed in nature and has been observed in plants, yeast, lactobacilli and in many animal tissues.

Fig. 25: Biosynthesis of flavin mononucleotide (FMN) and flavin dinucleotide (FAD) from riboflavin

Biosynthesis of Coenzyme A Coenzyme A is synthesized in the mammalian liver from the free vitamin pantothenic acid, which is required in the mammalian diet whereas plants and microorgnisms synthesize it. The five steps in the synthesis are shown in Fig. 26. The synthesis of coenzyme A starts with the phosphorylation of the vitamin pantothenic acid to produce 4'-phosphopantothenic acid. This reaction is catalyzed by pantothenate kinase. In the next step a peptide bond is formed between the amino group of cysteine and the carboxyl group of 4'- phosphopantothenic acid catalyzed by phosphopantothenylcysteine synthase to yield 4'- phosphopantothenylcysteine. The carboxyl group of the cysteine moiety is removed by phosphopantothenyl -cysteine decarboxylase forming 4'-phosphopantotheine. The transfer of an AMP moiety from ATP catalyzed by a pantotheninephosphate adenyltransferase forms dephospho-CoA. In the last step, a phosphate group is introduced into the 3'- hydroxyl group of the adenosine portion of dephospho-CoA by dephospho-CoA kinase to yield complete coenzyme A molecule.

Fig.26: Biosynthesis of Coenzyme A

Suggested Reading 1. Principles of Biochemistry by Lehninger 2. Biochemistry by L. Stryer