Reginald H. GarrettCharles M. Grisham

Chapter 26Synthesis and Degradation of

Nucleotides

Outline

• Nucleotide synthesis.• Purine synthesis.• Purine salvage pathway.• Purines degradation.• Pyrimidine synthesis.• Pyrimidines degradation.• Synthesis of deoxyribonucleotides that are

necessary for DNA synthesis.• Thymine nucleotide synthesis.

26.1 Can Cells Synthesize Nucleotides?

• Nearly all organisms synthesize purines and pyrimidines de novo (“anew”).

• Many organisms also "salvage" purines and pyrimidines from diet and degradative pathways.

• Ribose generates energy, but purine and pyrimidine rings do not.

• Nucleotide synthesis pathways are good targets for anti-cancer/antibacterial strategies.

26.2 Purine Synthesis

John Buchanan (1948) "traced" the sources of all nine atoms of the purine ring

• N-1 from aspartic acid. • N-3, N-9 from glutamine. • C-4, C-5, N-7 from glycine.

• C-6 from CO2.

• C-2, C-8 from THF - one carbon units.

Figure 26.2 The metabolic origin of the nine atoms in the purine ring system.

26.2 Purine Synthesis

26.2 Purines is a Waste Product in Birds

Figure 26.1 Nitrogen waste is excreted by birds principally as the purine analog, uric acid.

The purine ring is built on a ribose-5-P foundation

• Step 1: Ribose-5-P must be activated - by PPi.

• Phosphoribosyl pyrophosphate (PRPP), made by PRPP synthetase, is the limiting substance for purine synthesis. (Used in Trp and His synthesis.)

• Since PRPP is a branch point, the next step is the committed step - Gln PRPP amidotransferase.

• Step 2: Note the change in C-1 configuration. • G- and A-nucleotides inhibit this step - but at

separate sites.

26.2 Purine Synthesis, Steps 1 & 2

Purine Synthesis

Figure 26.3 The de novo pathway for purine synthesis.The first purine synthesized is inosine monophos. (IMP).IMP serves as a precursor to AMP and GMP.

Purine Synthesis, Steps 3-5

• Step 3: Glycine carboxyl condenses with amine of 5-phosphoribosyl-β-amine in two steps. • Glycine carboxyl activated by phosphorylation

from ATP. • Then the amine attacks the glycine carboxyl.

• Step 4: Formyl group of N10-formyl-THF is transferred to free amino group of GAR to form α-N-formylglycinamide ribonucleotide.

• Step 5: C-4 carbonyl forms a P-ester from ATP then active NH3 attacks C-4 to form an imine, namely formylglycinamidine ribonucleotide (FGAM).

Purine Synthesis

Figure 26.3 The de novo pathway for purine synthesis.

Purine Synthesis, Steps 6-8

Closure of the first ring, carboxylation and attack by aspartate

• Step 6: Similar in some ways to step 5. ATP activates the formyl group by phosphorylation, facilitating attack by N.

• Step 7: Carboxylation probably involves electron "push" from the amino group.

• Step 8: Attack by the amino group of aspartate links this amino acid with the carboxyl group.

Purine Synthesis

Figure 26.3 The de novo pathway for purine synthesis.

Purine Synthesis, Steps 9-11

Loss of fumarate, another 1-C unit and the second ring closure

• Step 9: Deprotonation of Asp-CH2 leads to cleavage to form fumarate.

• Step 10: Another 1-C addition catalyzed by THF. • Step 11: Amino group attacks formyl group to

close the second ring. • Note that 6 steps use ATP, but that this is really

seven ATP equivalents used to make IMP.

Purine Synthesis

Figure 26.3 This completes the de novo pathway for purine synthesis. IMP is the first purine made and serves as a precursor to AMP and GMP.

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

AMP and GMP are Synthesized from IMP

Reciprocal control occurs in two ways - see Figures 26.5 and 26.6

• GTP is the energy source for AMP synthesis, and ATP is the energy source for GMP.

• AMP is made by N addition from aspartate (in the familiar way).

• GMP is made by oxidation at C-2, followed by replacement of the O by N (from Gln).

• Last step of GMP synthesis is identical to the first two steps of IMP synthesis.

AMP is Made by Adenylosuccinate Lyase

AMP is made from IMP in two steps.

The first step converts IMP to adenylosuccinate and requires GTP.

The second step, catalyzed by adenylosuccinate lyase produces AMP, as shown in Figure 26.5.

AMP synthesis from IMP requires one additional ATP equivalent for a total of eight ATPs.

Figure 26.5 The synthesis of AMP and GMP from IMP.

AMP and GMP are Synthesized from IMP

GMP is Made by GMP Synthetase

GMP is made from IMP in two steps.

The first step requires oxidation at C-2 of the purine ring.

The second step, catalyzed by GMP synthetase, is a glutamine-dependent amidotransferase reaction that replaces the oxygen on C-2 with an amino group to yield 2-amino, 6-oxy purine nucleoside monophosphate – i.e., GMP.

GMP synthesis from IMP requires two additional ATP equivalents for a total of nine ATPs.

Formation of NDPs and NTPs

AMP and GMP are converted to NDPs using ATP and nucleoside monophosphate kinase.

ATP + GMP ADP + GDP

Conversion of GDP to GTP uses ATP and nucleoside diphosphate kinase.

ATP + GDP ADP + GTP

Regulation of the Purine Pathway

Figure 26.6 The regulatory circuit controlling purine biosynthesis. Allosteric regulation occurs in the first two steps, and AMP and GMP are competitive inhibitors in the two branches at right.

26.3 Purine Salvage Pathways

• Nucleic acid turnover (synthesis and degradation) is an ongoing process in most cells.

• Salvage pathways collect hypoxanthine and guanine and recombine them with PRPP to form nucleotides in the HGPRT reaction.

(Hypoxanthine-guanine phosphoribosyltranferase).• In L-N, purine synthesis is increased 200-fold and

uric acid is elevated in blood. • This increase may be due to PRPP feed-forward

activation of de novo pathways.

HGPRT Converts Bases Back to Nucleotides Using PRPP

Figure 26.7 Purine salvage by the HGPRT reaction. HGPRT salvage of hypoxanthine. SN2 gives a β-link.

Salvage pathways recover purine bases to use for resynthesis of nucleotides. e.g. the HGPRT reaction will recover hypoxanthine or guanine.

HGPRT Converts Bases Back to Nucleotides Using PRPP

Figure 26.7 Purine salvage by the HGPRT reaction. HGPRT salvage of guanine. SN2 gives a β-link.

Salvage pathways are very useful because of the high energy cost for denovo synthesis of nitrogen bases. The salvage pathway for adenine recovery(adenine phosphoribosyltranferase) is not shown.

Some Commonly Used Enzymes

• Nucleotidases cleave Pi from a nucleotide.• Nucleosidases cleave the base from a nucleoside. • Nucleoside phosphorylase cleaves the base from a

nucleoside using Pi. • Nucleoside kinase adds phosphate to a nucleoside.

N

NN

N

OCH2O - P -

NH2

OHHO

O

O

O

nucleoside phosphorylase

nucleotidase

nucleosidase_

_

O

Pi

26.4 Purine Catabolism

Purine catabolism leads to uric acid (see Figure 26.8)

• Nucleotidases and nucleosidases release ribose and phosphates and leave free bases.

• Xanthine oxidase and guanine deaminase route everything to xanthine.

• Xanthine oxidase converts xanthine to uric acid. • Note that xanthine oxidase can oxidize two

different sites on the purine ring system.

The Major Pathways of Purine Catabolism Lead to Uric Acid

• Nucleotides are first converted to nucleosides (loss of Pi) by intracellular nucleotidases.

• These nucleotidases are under strict regulation to ensure that their substrates (which are intermediates in many processes) are not depleted below critical levels.

• Nucleosides are then degraded by purine nucleoside phosphorylase (PNP) giving ribose-1-P and a nitrogen base.

• PNP products are converted to xanthine by guanine deaminase and xanthine oxidase.

The Major Pathways of Purine Catabolism Lead to Uric Acid

Figure 26.8 The major pathways for purine catabolism.

The Major Pathways of Purine Catabolism Lead to Uric Acid

Figure 26. The major pathways for purine catabolism.

The Purine Nucleotide Cycle in Skeletal Muscle Serves as an Anaplerotic Pathway

• Deamination of AMP to IMP by AMP deaminase, followed by resynthesis of AMP from IMP in de novo purine pathway enzymes, constitutes a purine nucleoside cycle (Figure 26.9).

• This cycle has the net effect of converting aspartate to fumarate plus NH4

+.

• This nucleotide cycle is important – fumarate that is generated replenishes the levels of citric acid intermediates lost in amphibolic side reactions.

• Skeletal muscle lacks the usual anaplerotic enzymes and relies on AMP deaminase for this purpose.

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

The Purine Nucleotide Cycle in Skeletal Muscle Serves as an Anaplerotic Pathway

Gout is a Disease Caused by an Excess of Uric Acid

• Xanthine oxidase (XO) in liver, intestines (and milk) can oxidize hypoxanthine (twice) 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 XO, is a treatment.

Figure 26.10 Xanthine oxidase catalyzes a hydroxylase-type reaction.

Gout is a Disease Caused by an Excess of Uric Acid

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

Allopurinol binds tightly to xanthine oxidase, preventing uric acid formation. Hypoxanthine and xanthine do not accumulate to harmful concentrations because they are more soluble and thus more easily excreted.

Gout is a Disease Caused by an Excess of Uric Acid

Animals Oxidize Uric Acid to Different Excretory Products

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

Primates, like birds, reptiles, and insects, excrete uric acid directly. Other animals use other routes of excretion.

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

Primates, like birds, reptiles, and insects, excrete uric acid directly. Other animals use other routes of excretion.

Animals Oxidize Uric Acid to Different Excretory Products

26.5 Pyrimidine Synthesis

• In contrast to purines, pyrimidines are not synthesized as nucleotides.

• Rather, the pyrimidine ring is completed before a ribose-5-P is added.

• Carbamoyl-phosphate and aspartate are the precursors of the six atoms of the pyrimidine ring.

• Mammals have two enzymes for carbamoyl phosphate synthesis – carbamoyl phosphate for pyrimidine synthesis is formed by carbamoyl phosphate synthetase II (CPS-II), a cytosolic enzyme.

Figure 26.13 The metabolic origin of the six atoms of the pyrimidine ring. Only two precursors contribute atoms to the six-membered pyrimidine ring.

26.5 Pyrimidine Synthesis

Carbamoyl Phosphate Synthetase II

• Step1: 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, 2 ATP.

• See Figure 27.14.• Mammalian CPS II is the committed step in

pyrimidine synthesis, because carbamoyl phosphate made by CPS II has no fate other than incorporation into pyrimidines.

Figure 26.14 The carbamoyl phosphate synthetase II (CPS II) reaction. This is the committed step in pyrimidine synthesis in mammals.

Carbamoyl Phosphate Synthetase II

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

Carbamoyl Phosphate Synthetase II

Step 2: Aspartate transcarbamoylase

• Step 2: Aspartate transcarbamoylase (ATCase) catalyzes the condensation of carbamoyl phosphate with aspartate to form carbamoyl-aspartate.

• Note that carbamoyl phosphate represents an ‘activated’ carbamoyl group.

• Aspartate transcarbamoylase is a classic two-state allosteric enzyme (ATP+, CTP-).

ATCase Converts Carbamoyl Phosphate to Carbamoyl Aspartate

Figure 26.15 The de novo pyrimidine biosynthetic pathway.

Steps 3-6 of Pyrimidine Biosynthesis

• Step 3: ring closure and dehydration - catalyzed by dihydroorotase.

• Step 4: Synthesis of a true pyrimidine (orotate) by DHO dehydrogenase.

• Step 5: Orotate is joined with a ribose-P to form orotidine-5'-phosphate.

• The ribose-P donor is PRPP.• Step 6: OMP decarboxylase makes UMP.These are six separate enzymes in procaryotes

and three multifunctional proteins in eucaryotes.

Metabolic Channeling by Multifunctional Enzymes of Pyrimidine Biosynthesis

• Eukaryotic pyrimidine synthesis involves metabolic channeling (the transfer of metabolites between different enzymatic sites on a multifunctional polypeptide).

• ATP requirements for denovo pyrimidine synthesis:• To UMP 4 ATP• To CTP 5 ATP (not counting UDP & UTP)

• CTP synthetase then forms CTP from UTP and ATP.

UMP Synthesis Leads to Formation of UTP and CTP

Figure 26.16(a) CTP synthesis from UTP. UDP is formed from UMP via an ATP-dependent nucleoside monophosphate kinase: UMP + ATP → UDP + ADPThen UTP is formed by nucleoside diphosphate kinase:

UDP + ATP → UTP + ADP

Pyrimidine Biosynthesis is Regulated at ATCase in Bacteria and at CPS II in Animals

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

26.3 Pyrimidine Salvage Pathways

PYRIMIDINES:

uracil

Uracil + PRPP --------------> UMP + PPi phosphoribosyl transferase

Orotate orotate

Uracil + PRPP ----------------> PyMP + PPi

Cytosine phosphoribosyl transferase

26.6 Pyrimidines Degradation

• In some organisms, free pyrimidines are salvaged and recycled to form nucleotides via phosphoribosyltransferase reactions.

• In humans, however, pyrimidines are recycled from nucleosides, but free pyrimidine bases are not salvaged.

• Catabolism of cytosine and uracil yields -alanine, ammonium ion, and CO2.

• Catabolism of thymine yields -aminoisobutyric acid, ammonium ion, and CO2.

Figure 26.18 Pyrimidine degradation.

Catabolism of cytosine and uracil yields -alanine, ammonium ion, and CO2.

Catabolism of thymine yields -aminoisobutyric acid, ammonium ion, and CO2.

26.6 Pyrimidines Degradation

26.7 Synthesis of Deoxyribonucleotides

• Reduction of an NDP at the 2'-position by replacement of the 2'-OH with hydride commits nucleotides to DNA synthesis.

• This reduction with hydride is catalyzed by ribonucleotide reductase.

• Ribonucleotide reductase is an 22-type enzyme - subunits R1 (86 kD) and R2 (43.5 kD).

• Each R1 subunit has regulatory site, a specificity site and an overall activity site. Each R2 subunit has part of the activity site.

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

26.7 Synthesis of Deoxyribonucleotides

E. Coli Ribonucleotide Reductase Has Three Different Nucleotide-Binding Sites

Figure 26.20 E. coli ribonucleotide reductase.

Ribonucleotide Reductase is Regulated by Nucleotide Binding• Ribonucleotide reductase is modulated in two

ways to maintain a balance of dATP, dGTP, dCTP, and dTTP.

• First, the catalytic activity of the enzyme of the enzyme must be turned on and off in response to need for dNTPs.

• Second, the amounts of each NDP substrate transformed must be controlled.

• ATP activates, dATP inhibits at the overall activity site.

• ATP, dATP, dTTP and dGTP bind at the specificity site to regulate the selection of substrates and the products made.

E. Coli Ribonucleotide Reductase

Effects of binding at S, A and C:

Bound at A Bound at S Accepted at CATP (on) ATP or dATP CDP or UDP

ATP (on) dTTP GDP favored CDP or UDP (no)

ATP (on) dGTP ADP favoredCDP, UDP, GDP

(no)

dATP (off) Any No reaction

Ribonucleotide Reductase is Regulated by Nucleotide Binding

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

E. Coli Ribonucleotide Reductase Has Three Different Nucleotide-Binding Sites

• Activity depends on Cys439, Cys225, and Cys462 on R1 and on Tyr122 on R2.

• Cys439 removes 3'-H, and dehydration follows, with disulfide formation between Cys225 and Cys462.

• The net result is hydride transfer to C-2‘.• Thioredoxin and thioredoxin reductase deliver

reducing equivalents.

Ribonucleotide Reductase Uses a Free Radical Mechanism

Figure 26.21 The free radical mechanism of ribonucleotide reduction. Ha designates the C-3' hydrogen and Hb the C-2' hydrogen atom.

Thioredoxin Reductase Mediates NADPH-Dependent Reduction of Thioredoxin

Figure 26.22(a) The oxidation-reduction cycle involving ribonucleotide reductase, thioredoxin, thioredoxin reductase, and NADPH. Thioredoxin functions in a number of metabolic roles besides deoxyribonucleotide synthesis.

26.8 Synthesis of Thymine Nucleotides

• Thymine nucleotides are made from dUMP, which derives from dUDP, dCDP.

• dUDPdUTPdUMPdTMP.• dCDPdCMPdUMPdTMP.

• Thymidylate synthase methylates dUMP at 5-position to make dTMP.

• N5,N10-methylene THF is 1-C donor.• Note role of 5-FU in chemotherapy.

Figure 26.24 Pathways of dTMP synthesis. dTMP production is dependent on dUMP formation from dCDP and dUDP.

Interestingly, formation of dUMP from dUDP passes through dUTP, which is then cleaved by dUTPase, a pyrophosphatase that removes PPi from dUTP. The action of dUTPase prevents dUTP from serving as a substrate in DNA synthesis.

26.8 Synthesis of Thymine Nucleotides

dCMP Deaminase Provides an Alternative Route to dUMP

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.

26.8 Synthesis of Thymine Nucleotides

Figure 26.26(a) The thymidylate synthase reaction.

Thymidylate Synthase

End Chapter 26Synthesis and Degradation of

Nucleotides