Chapter 26 The Synthesis and Degradation of Nucleotides Biochemistry by Reginald Garrett and Charles Grisham

Outline Can Cells Synthesize Nucleotides? How Do Cells Synthesize Purines? Can Cells Salvage Purines? How Are Purines Degraded? How Do Cells Synthesize Pyrimidines? How Are Pyrimidines Degraded? How Do Cells Form the Deoxyribonucleotides That Are Necessary for DNA Synthesis? How Are Thymine Nucleotides Synthesized?

Nucleotides and Nucleic Acids Nucleotides: (nucleoside + phosphate) Biological molecules that possess a heterocyclic nitrogenous base, a five-carbon sugar (ribose), and phosphate as principal components (chapter 10) Participate as essential intermediates in cellular metabolisms—NAD, FAD, ATP, cAMP… The elements of heredity and the agents of genetic information transfer—nucleic acids Nucleic acids: Nucleotides are the monomeric units of nucleic acid Two basic kinds of nucleic acids Deoxyribonucleic acid (DNA) Ribonucleic acid (RNA)

26.1 – Can Cells Synthesize Nucleotides? Nearly all organisms synthesize purines and pyrimidines "de novo biosynthesis pathway“ Many organisms also "salvage" purines and pyrimidines from diet and degradative pathways Ribose can be catabolized to generate energy, but nitrogenous bases do not Nucleotide synthesis pathways are good targets for anti-cancer/antibacterial strategies

26.2 – How Do Cells Synthesize Purines? John Buchanan (1948) "traced" the sources of all nine atoms of purine ring N-1: aspartic acid N-3, N-9: glutamine C-2, C-8: N10-formyl-THF - one carbon units C-4, C-5, N-7: glycine C-6: CO2

Figure 26.3 The de novo pathway for purine synthesis. Step 1: Ribose-5-phosphate pyrophosphokinase. Step 2: Glutamine phosphoribosyl pyrophosphate amidotransferase. Step 3: Glycinamide ribonucleotide (GAR) synthetase. Step 4: GAR transformylase. Step 5: FGAM synthetase (FGAR amidotransferase). Step 6: FGAM cyclase (AIR synthetase). Step 7: AIR carboxylase. Step 8: SAICAR synthetase. Step 9: adenylosuccinase. Step 10: AICAR transformylase. Step 11: IMP synthase.

IMP Biosynthesis IMP (inosinic acid or inosine monophosphate) is the immediate precursor to GMP and AMP First step: Ribose-5-phosphate pyrophosphokinase PRPP synthesis from ribose-5-phosphate and ATP PRPP is limiting substance for purine synthesis But PRPP is a branch point so next step is the committed step (fig 26.6) Second step: Gln PRPP amidotransferase Form phosphoribosyl-b-amine; Changes C-1 configuration (a→b) GMP and AMP inhibit this step - but at distinct sites Azaserine - Glutamine analog - inhibitor/anti-tumor

Figure 26. 4 The structure of azaserine Figure 26.4 The structure of azaserine. Azaserine acts as an irreversible inhibitor of glutamine-dependent enzymes by covalently attaching to nucleophilic groups in the glutamine-binding site.

Step 3: Glycinamide ribonucleotide (GAR) synthetase Glycine carboxyl condenses with amine in two steps Glycine carboxyl activated by -P from ATP Amine attacks glycine carboxyl Synthesize glycinamide ribonucleotide Step 4: Glycinamide ribonucleotide (GAR) transformylase Formyl group of N10-formyl-THF is transferred to free amino group of GAR Yield N-Formylglycinamide ribonucleotide

Step 5: Formylglycinamide ribonucleotide (FGAR) amidotransferase (FGAM synthetase) Formylglycinamidine ribonucleotide (FGAM) C-4 carbonyl forms a P-ester from ATP and active NH3 attacks C-4 to form imine Irreversibly inactivated by azaserine

Closure of the first ring, carboxylation and attack by aspartate Step 6: FGAM cyclase (AIR synthetase) Produce aminoimidazole nucleotide (AIR) Similar in some ways to step 5. ATP activates the formyl group by phosphorylation, facilitating attack by N. In avian liver, the enzymes for step 3, 4, and 6 (GAR synthetase, GAR transformylase, and AIR synthetase) reside on a polypeptide

Step 8: SAICAR synthetase Step 7: AIR carboxylase The product is carboxyaminoimidazole ribonucleotide (CAIR) Carbon dioxide is added in ATP-dependent reaction Step 8: SAICAR synthetase N-succinylo-5-aminoimidazole-4-carboxamide ribonucleotide Attack by the amino group of aspartate links this amino acid with the carboxyl group The enzymes for steps 7 and 8 reside on a bifunctional polypeptide in avian

Step 9: Adenylosuccinase (also see Fig 26.5) The product is 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR); remove fumarate AICAR is also an intermediate in the histidine biosynthetic pathway Step 10: AICAR transformylase N-formylaminoimidazole-4-carboxamide ribonucleotide (FAICAR) Another 1-C addition (N10-formyl-THF) Step 11: IMP synthase (IMP cyclohydrolase) Amino group attacks formyl group to close the second ring The enzymes for steps 10 and 11 reside on a bifunctional polypeptide in avian

6 ATPs are required in the purine biosynthesis from ribose-5-phosphate to IMP, but that this is really 7 ATP equivalents The dependence of purine biosynthesis on THF (tetrahydrofolate) in two steps means that methotrexate and sulfonamides block purine synthesis

Tetrahydrofolate and One-Carbon Units Folic acid, a B vitamin found in green plants, fresh fruits, yeast, and liver, is named from folium, Latin for “leaf”. Folates are acceptors and donors of one-carbon units for all oxidation levels of carbon except CO2 (for which biotin is the relevant carrier). The active form is tetrahydrofolate.

Tetrahydrofolate and One-Carbon Units Folates are acceptors and donors of one-carbon units for all oxidation levels of carbon except CO2 (for which biotin is the relevant carrier).

Tetrahydrofolate and One-Carbon Units Oxidation numbers are calculated by assigning valence bond electrons to the more electronegative atom and then counting the charge on the quasi ion. A carbon assigned four valence electrons would have an oxidation number of 0. The carbon in N5-methyl-THF (top left) is assigned six electrons from the three C-H bonds and thus has a oxidation number of -2.

Folate Analogs as Antimicrobial and Anticancer Agents De novo purine biosynthesis depends on folic acid compounds at steps 4 and 10 For this reason, antagonists of folic acid metabolism indirectly inhibit purine formation and, in turn, nucleic acid synthesis, cell growth, and cell development Rapidly growing cells, such as infective bacteria and fast-growing tumors, are more susceptible to such agents Sulfonamides are effective anti-bacterial agents Methotrexate and aminopterin are folic acid analogs that have been used in cancer chemotherapy

Sulfa drugs, or sulfonamides, owe their antibiotic properties to their similarity to p-aminobenzoate (PABA),an important precursor in folic acid synthesis. Sulfonamides block folic acid formation by competing with PABA.

AMP and GMP are Synthesized from IMP Figure 26.5 The synthesis of AMP and GMP from IMP.

AMP and GMP are synthesized from IMP IMP is the precursor to both AMP and GMP AMP synthesis Step 1: Adenylosuccinate synthetase The 6-O of inosine is displaced by aspartate to yield adenylosuccinate GTP is the energy input for AMP synthesis, whereas ATP is energy input for GMP Step 2: Adenylosuccinase (adenylosuccinate lyase) Carries out the nonhydrolytic removal of fumarate from adenylosuccinate, leaving AMP. The same enzyme catalyzing Step 9 in the purine pathway

Starting from ribose-5-phosphate GTP synthesis Step 1: IMP dehydrogenase Oxidation at C-2 NAD+-dependent oxidation xanthosine monophosphate (XMP) Step 2: GMP synthetase Replacement of the O by N (from Gln) ATP-dependent reaction; PPi Starting from ribose-5-phosphate 8 ATP equivalents are consumed in the AMP synthesis 9 ATP equivalents in GMP synthesis

The regulation of purine synthesis Reciprocal control occurs in two ways IMP synthesis: Allosterically regulated at the first two steps R-5-P pyrophosphokinase: ADP & GDP Phosphoribosyl pyrophosphate amidotransferase A “series”: AMP, ADP, and ATP G “series”: GMP, GDP, and GTP PRPP is “feed-forward” activator

AMP synthesis: adenylosuccinate synthetase is feedback-inhibited by AMP GMP synthesis: IMP dehydrogenase is feedback-inhibited by GMP

Nucleoside diphosphate and triphosphate Nucleoside diphosphate: ATP-dependent kinase Adenylate kinase: AMP +ATP → ADP +ADP Guanylate kinase: GMP +ATP → GDP +ADP Nucleoside triphosphate: non-specific enzyme Nucleoside diphosphate kinase GDP +ATP  GTP +ADP NDP +ATP  NTP +ADP (N=G, C, U, and T)

26.3 – Can Cells Salvage Purines? Salvage pathways Nucleic acid turnover (synthesis and degradation) is an ongoing metabolic process mRNA in particular is actively synthesized and degraded Lead to release of free purines; adenine, guanine, and hypoxanthine (the base in IMP; Fig 26.8) Salvage pathways exist to recover them in useful form Involve resynthesis of nucleotides from bases via phosphoribosyltransferases (PRT)

26.3 – Can Cells Salvage Purines? Base + PRPP Nucleoside-5’-phosphate + PPi The purine phosphoribosyltransferases are adenine phosphoribosyltransferases (APRT) and hypoxanthine-guanine phosphoribosyltransferases (HGPRT) Collect hypoxanthine and guanine and recombine them with PRPP to form nucleotides in the HGPRT reaction (Fig 26.7) Absence of HGPRT is cause of Lesch-Nyhan syndrome (sex-linked); In Lesch-Nyhan, purine synthesis is increased 200-fold and uric acid is elevated in blood

Hyperxanthine-Guanine PhosphoRibosylTransferase Figure 26.7 Purine salvage by the HGPRT reaction.

Victims of Lesch-Nyhan syndrome experience severe arthritis due to accumulation of uric acid, as well as retardation, and other neurological symptoms.

26.4 – How Are Purines Degraded? Purine catabolism leads to uric acid Nucleotidases and nucleosidases release ribose and phosphates and leave free bases Nucleotidase: NMP + H2O → nucleoside + Pi Nucleosidase: nucleoside + H2O → base + ribose PNP: nucleoside + Pi → base + ribose-P The PNP products are converted to xanthine by xanthine oxidase and guanine deaminase Xanthine oxidase converts xanthine to uric acid Note that xanthine oxidase can oxidize two different sites on the purine ring system Neither adenosine nor deoxyadenosine is a substrate for PNP Converted to inosine by adenosine deaminase (ADA)

Figure 26. 8 The major pathways for purine catabolism in animals Figure 26.8 The major pathways for purine catabolism in animals. Catabolism of the different purine nucleotides converges in the formation of uric acid.

Severe combined immunodeficiency syndrome (SCID) The effect of elevated levels of deoxyadenosine on purine metabolism. If ADA is deficient or absent, deoxyadenosine is not converted into deoxyinosine as normal (see Figure 26.8). Instead, it is salvaged by a nucleoside kinase, which converts it to dAMP, leading to accumulation of dATP and inhibition of deoxynucleotide synthesis (see Figure 26.24). Thus, DNA replication is stalled.

Convert aspartate to fumarate plus NH4+ The purine nucleoside cycle in skeletal muscle Serve as an anaplerotic pathway Convert aspartate to fumarate plus NH4+ Figure 26.9 The purine nucleoside cycle for anaplerotic replenishment of citric acid cycle intermediates in skeletal muscle.

Xanthine Oxidase and Gout Xanthine Oxidase in liver, intestines mucosa, and milk can oxidize hypoxanthine to xanthine and xanthine to uric acid Humans and other primates excrete uric acid in the urine, but most N goes out as urea Birds, reptiles and insects excrete uric acid and for them it is the major nitrogen excretory compound Gout occurs from accumulation of uric acid crystals in the extremities Allopurinol, which inhibits xanthine oxidase , is a treatment

Figure 26.11 Allopurinol, an analog of hypoxanthine, is a potent inhibitor of xanthine oxidase. Figure 26.10 Xanthine oxidase catalyzes a hydroxylase-type reaction.

Animals other than humans oxidize uric acid to form excretory products Urate oxidase: Allantoin Allantoinase: Allantoic acid Allantoicase: Urea Urease: Ammonia Figure 26.12 The catabolism of uric acid to allantoin, allantoic acid, urea, or ammonia in various animals.

26.5 – How Do Cells Synthesize Pyrimidines? In contrast to purines, pyrimidines are not synthesized as nucleotides The pyrimidine ring is completed before a ribose-5-P is added Carbamoyl-P and aspartate are the precursors of the six atoms of the pyrimidine ring

Figure 26.15 The de novo pyrimidine biosynthetic pathway.

de novo Pyrimidine Synthesis Step 1: Carbamoyl Phosphate synthesis Carbamoyl phosphate for pyrimidine synthesis is made by carbamoyl phosphate synthetase II (CPS II) This is a cytosolic enzyme (whereas CPS I is mitochondrial and used for the urea cycle) Substrates are HCO3-, glutamine (not NH4+), 2 ATP In mammals, CPS-II can be viewed as the committed step in pyrimidine synthesis Bacteria have but one CPS; thus, the committed step is the next reaction, which is mediated by aspartate transcarbamoylase (ATCase)

(also called carbonyl-phosphate) Figure 26.14 The reaction catalyzed by carbamoyl phosphate synthetase II (CPS II).

Step 2: Aspartate transcarbamoylase (ATCase) catalyzes the condensation of carbamoyl phosphate with aspartate to form carbamoyl-aspartate carbamoyl phosphate represents an ‘activated’ carbamoyl group Step 3: dihydroorotase ring closure and dehydration via intramolecular condensation Produce dihydroorotate

Step 4: dihydroorotate dehydrogenase Synthesis of a true pyrimidine (orotate) Step 5: orotate phosphoribosyltransferase Orotate is joined with a ribose-P to form orotidine-5’-phosphate (OMP) The ribose-P donor is PRPP Step 6: OMP decarboxylase OMP decarboxylase makes UMP (uridine-5’-monophposphate, uridylic acid)

Metabolic channeling In bacteria, the six enzymes are distinct Eukaryotic pyrimidine synthesis involves channeling and multifunctional polypeptides CPS-II, ATCase, and dihydroorotase are on a cytosolic polypeptide Orotate PRT and OMP decarboxylase on the other cytosolic polypeptide (UMP synthase) The metabolic channeling is more efficient

UTP and CTP Nucleoside monophosphate kinase UMP + ATP → UDP + ADP Nucleoside diphosphate kinase UDP + ATP → UTP + ADP CTP sythetase forms CTP from UTP and ATP

Regulation of pyrimidine biosynthesis In bacteria allosterically inhibited at ATCase by CTP (or UTP) allosterically activated at ATCase by ATP (compete with CTP) In animals UDP and UTP are feedback inhibitors of CPS II PRPP and ATP are allosteric activators

Figure 26.17 A comparison of the regulatory circuits that control pyrimidine synthesis in E. coli and animals.

26.6 – How Are Pyrimidines Degraded? In some organisms, free pyrimidines are salvaged and recycled to form nucleotides In humans, pyrimidines are recycled from nucleosides, but free pyrimidine bases are not salvaged Catabolism of cytosine and uracil yields -alanine, ammonium, and CO2 -alanine can be recycled into the synthesis of coenzyme A Catabolism of thymine yields -aminoisobutyric acid, ammonium, and CO2

Figure 26.18 Pyrimidine degradation. Carbons 4, 5, and 6 plus N-1 are released as b-alanine, N-3 as NH4+, and C-2 as CO2. (The pyrimidine thymine yields b-aminoisobutyric acid.) Recall that aspartate was the source of N-1 and C-4, -5, and -6, while C-2 came from CO2 and N-3 from NH4+ via glutamine.

26.7 – How Do Cells Form the Deoxyribonucleotides That Are Necessary for DNA Synthesis? 90% of the total nucleic acid in cells is RNA, with the remainder being DNA The deoxynucleotides have only one metabolic purpose: to serve as precursor for DNA synthesis NDPs are the substrate for deoxynucleotides formation Reduction at 2’-position of ribose ring in NDPs produces 2’-deoxy forms of these nucleotides

Replacement of 2’-OH with hydride is catalyzed by ribonucleotide reductase Figure 26.19 Deoxyribonucleotide synthesis involves reduction at the 2'-position of the ribose ring of nucleoside diphosphates.

An 22-type enzyme has subunits R1 (2, 86 kD) and R2 (b2, 43.5 kD) R1 has two regulatory sites, a substrate specificity site (S) and an overall activity site (A) Figure 26.20 E. coli ribonucleotide reductase.

Ribonucleotide Reductase The enzyme system consists of 4 proteins Two of which constitute the Ribonucleotide Reductase (a2b2) Thioredoxin and thioredoxin reductase deliver reducing equivalents Has three different nucleotide-binding sites Substrate: NDPs (active site) Activity-determining: ATP & dATP (overall actiyity site) Specificity-determining: ATP, dTTP, dGTP, and dATP (sbustrate specific site)

Activity depends on Cys439, Cys225, and Cys462 on R1 and on Tyr122 on R2 (generate free radical) Tyr122 free radical on R2 leads to removal of the Ha hydrogen (Cys439) and creation of a C-3‘ radical Dehydration follows with disulfide formation between Cys225, and Cys462 and forms the dNDP product

Thioredoxin provides the reducing power for ribonucleotide reductase NADPH is the ultimate source Reversible Sulfide : sulfhydryl transition Figure 26.22 The (—S—S—)/(—SH HS—) oxidation-reduction cycle involving ribonucleotide reductase, thioredoxin, thioredoxin reductase, and NADPH.

Regulation of dNTP Synthesis The overall activity of ribonucleotide reductase must be regulated ATP activates, dATP inhibits at the overall activity site Balance of the four deoxynucleotides must be controlled ATP, dATP, dTTP and dGTP bind at the substrate specificity site to regulate the selection of substrates and the products made

Figure 26.23 Regulation of deoxynucleotide biosynthesis: The rationale for the various affinities displayed by the two nucleotide-binding regulatory sites on ribonucleotide reductase.

26.8 – How Are Thymine Nucleotides Synthesized? Thymine nucleotides are made from dUMP, which derives from dUDP, dCDP Thymidylate synthase methylates dUMP at 5-position to make dTMP N5, N10-methylene THF is 1-C donor If the dCDP pathway is traced from the common pyrimidine precursor, UMP, it will proceed as follows: dUTPase dCMP deaminase UMP → UDP→ UTP → CTP → CDP → dCDP → dCMP → dUMP → dTMP

Figure 26. 25 (a) The dCMP deaminase reaction Figure 26.25 (a) The dCMP deaminase reaction. An alternative route to dUMP is provided by dCDP, which is dephosphorylated to dCMP and then deaminated by dCMP deaminase

Synthesis of dTMP from dUMP is catalyzed by thymidylate synthase This enzyme methylates dUMP at the 5-position to create dTMP The methyl donor is the one-carbon folic acid derivative N5, N10-methylene-THF The reaction is a reductive methylation; the one-carbon unit is transferred at the methylene level of reduction and then reduced to the methyl level The THF cofactor is oxidized to yield DHF DHFR reduces DHF back to THF for serving again dTMP synthesis has become a preferred target for inhibitors designed to disrupt DNA synthesis

Figure 26.26 The thymidylate synthase reaction.

Precursors and analogs of folic acid employed as antimetabolites: sulfonamides (see Human Biochemistry box on page 896), as well as methotrexate, aminopterin, and trimethoprim, whose structures are shown here. These compounds shown here bind to dihydrofolate reductase (DHFR) with about 1000-fold greater affinity than DHF and thus act as virtually irreversible inhibitors.

Fluoro-substituted analogs as therapeutic agents The effect of the 5-fluoro substitution on the mechanism of action of thymidylate synthase. An enzyme thiol group (from a Cys side chain) ordinarily attacks the 6-position of dUMP so that C-5 can react as a carbanion with N5,N10-methylene-THF. Normally, free enzyme is regenerated following release of the hydrogen at C-5 as a proton. Because release of fluorine as F+ cannot occur, the ternary (three-part) complex of [enzyme: flourouridylate:methylene-THF] is stable and persists, preventing enzyme turnover. (The N5,N10-methylene-THF structure is given in abbreviated form.)

5-fluorouracil (5-FU) is used as a chemotherapeutic agent in the treatment of human cancers 5-fluorocytosine is used as an antifungal drug 5-fluoroorotate is an effective antimalarial drug