Chapter 26

The Synthesis and Degradation of Nucleotides

Biochemistry

by

Reginald Garrett and Charles Grisham

Outline1. Can Cells Synthesize Nucleotides?2. How Do Cells Synthesize Purines?3. Can Cells Salvage Purines?4. How Are Purines Degraded?5. How Do Cells Synthesize Pyrimidines?6. How Are Pyrimidines Degraded?7. How Do Cells Form the

Deoxyribonucleotides That Are Necessary for DNA Synthesis?

8. 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 acids1.Deoxyribonucleic acid (DNA)2.Ribonucleic acid (RNA)

26.1 – Can Cells Synthesize Nucleotides?

1. Nearly all organisms synthesize purines and pyrimidines "de novo biosynthesis pathway“

2. 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 BiosynthesisIMP (inosinic acid or inosine monophosphate) is

the immediate precursor to GMP and AMPFirst 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--amine; Changes C-1

configuration (→)– GMP and AMP inhibit this step - but at distinct sites – Azaserine - Glutamine analog - inhibitor/anti-tumor

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

1. Glycine carboxyl activated by -P from ATP 2. 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

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

Closure of the first ring, carboxylation and attack by aspartate

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.

Figure 26.5 The synthesis of AMP and GMP from IMP.

AMP and GMP are Synthesized from IMP

AMP and GMP are synthesized from IMP

IMP is the precursor to both AMP and GMP • AMP synthesisStep 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

• GTP synthesisStep 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 synthesisReciprocal control occurs in two ways

IMP synthesis:

Allosterically regulated at the first two steps

1. R-5-P pyrophosphokinase:

• ADP & GDP

2. 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

Figure 26.7 Purine salvage by the HGPRT reaction.

Hyperxanthine-Guanine PhosphoRibosylTransferase

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. Catabolism of the different purine nucleotides converges in the formation of uric acid.

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.

Severe combined immunodeficiency syndrome (SCID)

Figure 26.9 The purine nucleoside cycle for anaplerotic replenishment of citric acid cycle intermediates in skeletal muscle.

The purine nucleoside cycle in skeletal muscle Serve as an anaplerotic pathway

• Convert aspartate to fumarate plus NH4+

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 26.10 Xanthine oxidase catalyzes a hydroxylase-type reaction.

Figure 26.11 Allopurinol, an analog of hypoxanthine, is a potent inhibitor of xanthine oxidase.

Figure 26.12 The catabolism of uric acid to allantoin, allantoic acid, urea, or ammonia in various animals.

Animals other than humans oxidize uric acid to form excretory products

• Urate oxidase: Allantoin

• Allantoinase: Allantoic acid

• Allantoicase: Urea

• Urease: Ammonia

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)

Figure 26.14The reaction catalyzed by carbamoyl phosphate synthetase II (CPS II).

(also called carbonyl-phosphate)

• 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 kinaseUDP + 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 -alanine, N-3 as NH4+, and C-2 as

CO2. (The pyrimidine thymine yields -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

Figure 26.19 Deoxyribonucleotide synthesis involves reduction at the 2'-position of the ribose ring of nucleoside diphosphates.

• Replacement of 2’-OH with hydride is catalyzed by ribonucleotide reductase

Figure 26.20 E. coli ribonucleotide reductase.

• An 22-type enzyme has subunits R1 (2, 86 kD) and R2 (2, 43.5 kD)

• R1 has two regulatory sites, a substrate specificity site (S) and an overall activity site (A)

Ribonucleotide Reductase

• The enzyme system consists of 4 proteins– Two of which constitute the Ribonucleotide

Reductase (– 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

Figure 26.22 The (—S—S—)/(—SH HS—) oxidation-reduction cycle involving ribonucleotide reductase, thioredoxin, thioredoxin reductase, and NADPH.

• Thioredoxin provides the reducing power for ribonucleotide reductase

• NADPH is the ultimate source• Reversible Sulfide : sulfhydryl transition

Regulation of dNTP Synthesis

1. The overall activity of ribonucleotide reductase must be regulated

– ATP activates, dATP inhibits at the overall activity site

2. 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:

UMP → UDP→ UTP → CTP → CDP → dCDP → dCMP → dUMP → dTMP

dUTPase

dCMP deaminase

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.

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.)

• Fluoro-substituted analogs as therapeutic agents

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