L11 Nucleotide Metabolism

• 核苷酸代谢

一、Outlines of Pathways in Nucleotide Metabolism

Overview of nucleotide metabolism

• precursors to nucleic acid （RNA, DNA)；
• critical elements energy metabolism （ATP）；
• carriers of activated metabolites for biosynthesis (CDP-sugar)；
• structural moieties of coenzymes （FAD， NAD）；
• metabolic regulators and signal molecule (cAMP)

De novo pathways for synthesis and utilization of ribonucleotides (orange) and deoxyribonucleotides (blue).

Nucleotides arise through de novo synthesis from low-molecular-weight precursors or through salvage of nucleosides or bases.

Reutilization of purine and pyrimidine bases

The figure shows relationships between nucleic acid catabolism (blue) and resynthesis of nucleotides by salvage pathways (red).

• Involved Terms:

  • 核碱基（英语：Nucleobase）是指一类含氮碱基（nitrogenous base），在生物学上通常简单地称之碱基（base）。是在DNA和RNA中，起配对作用的部分。核碱基都是杂环化合物，其氮原子位于环上或取代氨基上，其中一部分（取代氨基，以及嘌呤环的1位氮、嘧啶环的3位氮）直接参与碱基配对。
  • 核苷（英语：Nucleoside）是一类糖甘胺（glycosylamine）分子，组成物是碱基加上环状核糖或脱氧核糖。例如胞苷、尿苷、腺苷、鸟苷与腺苷，这些核苷加上一个磷酸基团就是核苷酸，为DNA与RNA的组成单位。
  • A nucleotide is composed of three distinctive chemical sub-units: a five-carbon sugar molecule, a nitrogenous base—which two together are called a **nucleoside**—and one phosphate group. With all three joined, a nucleotide is also termed a “nucleoside monophosphate“, “nucleoside diphosphate“ or “nucleoside triphosphate“, depending on how many phosphates make up the phosphate group.
  • 

• De Novo（再次）: Most organisms can synthesize purine and pyrimidine nucleotides from low-molecule-weight precursors in amounts sufficient for their needs.

• Salvage: Most organisms can also synthesize nucleotides from nucleosides or bases.

The most common route for cleavage to the free base (nucleobase) involves the action of a nucleoside phosphorylase（核苷磷酸化酶）.

1. PRPP: A Central Metabolite in De Novo and Salvage Pathways

PRPP is an activated ribose-5-phosphate（核糖-5-磷酸） derivative used in both salvage and de novo pathways.

It is formed through the action of PRPP synthetase, which activates carbon 1 of ribose-5-phosphate by transferring to it the pyrophosphate moiety of ATP

A phosphoribosyltransferase（磷酸核糖转移酶） reaction catalyzes the reversible transfer of a free base to the ribose of PRPP, displacing pyrophosphate and producing a nucleoside monophosphate.

Because the deoxyribose（去氧核醣） analog of PRPP is absent from most cells, these enzymes are not involved directly in deoxyribonucleotide metabolism.

二、De Novo Biosynthesis of Purine Nucleotides

Low-molecular-weight precursors to the purine ring

The source of each atom in the ring, as established with isotopic tracer studies of uric acid synthesis.

From PRPP to IMP

The enzymes involved are:

1. PRPP amidotransferase 酰胺转移酶
2. GAR synthetase
3. GAR transformylase 转甲酰酶
4. FGAR amidotransferase
5. AIR synthetase
6. AIR carboxylase
7. SAICAR synthetase
8. Adenylosuccinate lyase
9. AICAR transformylase
10. IMP cyclohydrolase 环水解酶

Model of the purinosome（嘌呤体）

The figure shows a hypothetical model for a multi-enzyme complex in animal cells that transforms PRPP to IMP.

Serine hydroxymethyltransferase (SHMT，丝氨酸羟甲基转移酶) and the trifunctional enzyme C1-tetrahydrofolate (THF) synthase, also proposed to be part of the complex, channel one-carbon units from serine into 10-formyl-THF（10-甲酰基-THF） for the transformylase reactions.

IMP, the first fully formed purine nucleotide, is a branch point between adenine and guanine nucleotide biosynthesis.

• Inosine-5‘-monophosphate （IMP）：肌苷-5’-单磷酸

Pathways from IMP to GMP and AMP

Enzymes are as follows:

1. G-1 = IMP dehydrogenase
2. G-2 = GMP synthetase
3. A-1 = Adenylosuccinate synthetase
4. A-2 = Adenylosuccinate lyase

Nucleotides are active in metabolism primarily as the nucleoside triphosphates.

GMP and AMP are converted to their corresponding triphosphates through two successive phosphorylation reactions.

• Conversion to the diphosphates involves specific ATP-dependent kinases.

Nucleoside diphosphate kinase, an equilibrium-driven enzyme, transfers a phosphoryl group from ATP in the synthesis of all other nucleoside triphosphates.

Regulation of de novo purine biosynthesis

Utilization of Adenine Nucleotides in Coenzyme Biosynthesis

An important metabolic role of purine nucleotides is the synthesis of coenzymes, primarily those containing an adenylate moiety.

These include:

1. Flavin nucleotides 黄素核苷酸
2. Nicotinamide nucleotides 烟酰胺核苷酸
3. Coenzyme A

Purine Degradation and Formation of Uric Acid

• All purine degradation leads to uric acid.

1. Purine Degradation and Gout

Catabolism of uric acid to ammonia and CO2

Uric acid and its urate salts are quite insoluble.

1. Birds, reptiles, and insects: disposition of excess nitrogen that uses very little water: the waste material is excreted essentially as uric acid crystals;
2. Mammalians: difficulties, allantoin（尿囊素） by urate oxidase;
   • Humans and most primates: no urate oxidase→ high blood uric acid.
   • Uric acid is powerful scavenger of free radicals, providing protection against oxidative damage.

In North America and Europe, 3/1000 suffer from hyperuricemia-chronic elevation of blood uric acids well beyond the already high levels.

Prolonged or acute elevation of blood urate leads to its precipitation（沉淀,析出）, as crystals of sodium urate, resulting in a painful arthritis（关节炎）, which, if untreated, leads to ultimately to severe degeneration of the joints.

Eating and drinking purine-rich foods are apt to（适应） stimulate acute gouty attacks in susceptible individuals. Because such foods include “rich” items such as liver, sweetbreads, anchovies, and wine, gout is historically associated with an excess of high living.

Gout results either from overproduction of purine nucleotides, leading to uric acid synthesis, or from impaired uric acid excretion through the kidney.

Several known genetic alterations in purine metabolism can lead to purine oversynthesis, uric acid overproduction, and gout.

1. Elevated activity of PRPP synthetase: mutations cause the enzyme to become resistant to feedback inhibition by purine nucleotides. PRPP ↑➔ amidotransferase reaction↑ ➔ de novo purine biosynthesis↑ ➔ uric acid↑➔ gout;

2. Deficiency of the salvage enzyme hypoxanthine（次黄嘌呤）-guanine phosphoribosyltransferase (HGPRT) ↓or adenine phosphoribosyltransferase (APRT) ↓➔ consumes PRPP ↓➔ PRPP ↑ ➔ de novo purine biosynthesis↑ ➔uric acid↑➔gout;

3. Patients with glucose-6-phosphatase (G6P) deficiency are also gouty. PRPP is the key. G6Pase deficiency ↓➔ G6P accumulation in the liver ↑ ➔ pentose phosphate pathway (PPP) ↑ ➔ ribose-5-phosphate (R5P) ↑ ➔ increases PRPP ↑ ➔uric acid↑➔gout;

   Gout is also a consequence of cancer chemotherapy, presumably resulting from an overload of purine caused by nucleic acid degradation after death of tumor cells

Treatment of Gout: Allopurinol（别嘌呤醇）

Many cases of gout are successfully treated by the antimetabolite allopurinol, a structural analog of hypoxanthine in which the N7 and C8 positions are interchanged.

Xanthine dehydrogenase（黄嘌呤脱氢酶） hydroxylates allopurinol at C2 (as it does hypoxanthine), giving alloxanthine, which remains tightly bound to the reduced form of the enzyme.

Allopurinol is thus a suicide substrate that strongly inhibits xanthine dehydrogenase.

• This inhibition causes accumulation of hypoxanthine and xanthine, both of which are more soluble and, hence, more readily excreted than is uric acid.

Salvage Pathways of Purine Nucleotides

• hypoxanthine-guanine phosphoribosyltransferase (HGPRT)
• adenine phosphoribosyltransferase (APRT).

1. Salvage of Purines and Lesch-Nyhan Syndrome

“Null mutations”: total loss of the HGPRT enzyme;

In humans, as much as 90% of free purines are salvaged and reused, rather than degraded or excreted;

• If guanine and hypoxanthine are not salvaged, and are instead degraded by the pathway, leading to excess excretion of uric acid;
  • This is sex-linked recessive trait because the gene for HGPRT is located on the X chromosome;

Patients display a severe gouty arthritis, but they also have a dramatic malfunction of the nervous system, manifested as behavioural disorders, motor disability, learning disability, hostile or aggressive behavior, or often self-directed. In the most extreme cases, patients nibble at their fingertips or, if restrained, their lips, causing severe self-mutilation. “Nailbiting”;

• No successful treatment;
• Overproduction of uric acid can be blocked by allopurinol, but this treatment has no efficacy against the neurological features of Lesch-Nyhan syndrome;
• Can be identified by prenatally（产前的；胎儿期的；出生以前的） through amniocentesis (羊膜腔穿刺术).

Unexpected Consequence of Defective Purine Catabolism: Immunodeficiency

1. Metabolic consequences of adenosine deaminase (ADA) deficiency

AdoHcy = S-adenosylhomocysteine. S-腺苷同型半胱氨酸

Severe combined immunodeficiency disease (SCID): Patients with this condition are susceptible, often fatally, to infectious diseases because of a total inability to mount an immune response.

Both B and T lymphocytes are affected; neither class of cells can proliferate.

• Heritable lack of the degradative enzyme adenosine deamiase (ADA)

ADA also acts on 2’-deoxyadenosine, which arises from the degradation of DNA.

1. ADA deficiency ➔2’-deoxyadenosine↑ ➔ dATP↑ ➔ DNA synthesis↓ ➔ T and B cell proliferation↓ ➔ immune response↓; activation of ATP catabolism; activation of apoptosis
2. ADA deficiency ➔2’-deoxyadenosine↑ adenosine↑ ➔ AdoHcy hydrolase↓ ➔ AdoHcy↑ ➔ methyltransferase↓ ➔ methylation reactions↓

Standard treatment: frequent injections of purified bovine ADA covalent attached to the polymer polyethylene glycerol (PEG，聚甘油), which corrects the metabolic abnormalities, defects in the immune system usually persist.

• ADA deficiency was the first human disease to be treated by gene therapy.
  • In 1992, a four-year-old girl with this condition was treated with a viral vector expressing ADA. She is in her twenties, relatively healthy (also with ADA-PEG)

三、Pyrimidine Nucleotide Metabolism

De novo synthesis of pyrimidine nucleotides

Pyrimidine nucleotide synthesis occurs primarily at the free base level, with conversion to a nucleotide occurring late in the unbranched pathway.

• With only two precursors (aspartate and carbamoyl phosphate), providing all the C and N atoms of the pyrimidine ring)

Two major distinctions from the purine pathway

Enzyme Involved in above figure:

1. Carbamoyl phosphate synthetase
2. Aspartate transcarbamolyase
3. dihydroorotase
4. dihydroorotate dehydrogenase
5. orotate phosphoribosyltransferase
6. OMP decarboxylase
7. UMP kinase
8. nucleoside diphosphate kinase
9. CTP synthetase

• CTP is synthesized from UTP by a glutamine-dependent amidotransferase reaction, catalyzed by CTP synthetase.

1. the pyrimidine ring is assembled as a free base, with conversion to a nucleotide occurring later in the pathway, when the base orotic acid (乳清酸) is converted to orotidine monophosphate (奥洛替丁一磷酸), or OMP
2. The pyrimidine pathway is unbranched. Uridine triphosphate (UTP) is an end product of the pathway and is also the substrate for formation of cytidine triphosphate (CTP), the other end product.

Pyrimidine synthesis begins with formation of carbamoyl phosphate, a reaction catalyzed by carbamoyl phosphate synthetase（氨甲酰磷酸合成酶）.

• Carbamoyl phosphate is formed from ATP, bicarbonate, and the amide nitrogen from glutamine.

This amidotransferase reaction involves four separate chemical steps

Channeling in carbamoyl phosphate synthetase (CPS)

• 氨甲酰磷酸合成酶

1. Carbamoyl phosphate synthetase (CPS)

Bacterial CPS is a heterodimer composed a large subunit and a small subunit. The X-ray crystal structure of E.coli CPS, substrate binding sites and domains…

• Small subunit: functions as a glutaminase（谷氨酰胺酶）, catalyzing the hydrolysis of glutamine to form ammonia, which is delivered to the large subunit.
• Large subunit: two active sites: one binds bicarbonate, ATP, and ammonia and catalyzes carboxyphosphate and carbamate synthesis; the other active site binds to a second ATP and catalyzes carbamoyl phosphate formation.

The three unstable intermediates (ammonia, carboxyphosphate and carbamate) do not diffuse from the enzyme until converted to the final product, carbamoyl phosphate.

A 96 A tunnel in the interior of the enzyme through which the intermediates are channeled from one active site to the next (the dotted lines), a common feature in many enzymes and is one mechanism by which enzymes increase reaction efficiency and avoid unwanted side reactions with unstable intermediates….

2. Eukaryotic cells contains two forms of CPS

Form I: in mitochondria, has a preference for ammonia as substrate, and is used in arginine biosynthesis and the urea cycle.

Form II: in cytosol, has a strong preference for glutamine as nitrogen donor, for pyrimidine biosynthesis

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In eukaryotes the first three reactions of pyrimidine synthesis are catalyzed by a trifunctional enzyme, the CAD protein.

Similarly, the last two reactions are catalyzed by a bifunctional enzyme, UMP synthase.

• CAD: carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase： 氨甲酰磷酸合成酶2，天门冬氨酸转移酶，和二氢乳清酸酶
• Uridine monophosphate （UMP） synthetase： 尿苷酸合成酶

Miller syndrome （1 in 1 million ）: mutation in dihydroorotate dehydrogenase gene, developmental anomalies including cleft lip（唇裂；兔唇） or palate, absent digits（味觉缺失）, and ocular anomalies（眼部异常）. Miller syndrome is a rare condition that mainly affects the development of the face and limbs（四肢）.

Hereditary orotic aciduria: defects in UMP synthase gene, accumulate orotic acid, a block at the orotate phosphoribosyltransferase reaction

Catabolic pathways in pyrimidine nucleotide metabolism

四、Glutamine-Dependent Amidotransferases

A common theme in purine and pyrimidine biosynthesis. Five different reactions in these two pathways:

• PRPP amidotransferase
• FGAR amidotransferase
• GMP synthetase
• Carbamoyl phosphate synthetase
• CTP synthetase

The glutamine amido group (unreactive, non-nucleophilic) is activated by hydrolysis at the active site to deliver nucleophilic ammonia in high concentrations sufficient to aminate the specific acceptor (usually the carbonyl group of an amide). ATP serves as an energy input to force the equilibrium in the amination direction

五、Deoxyribonucleotide Biosynthesis and Metabolism

Overview of deoxyribonucleoside triphosphate (dNTP) biosynthesis

1. Structure of E. coli ribonucleoside diphosphate reductase

2. Reductive electron transport sequences in the action of rNDP Reductase

Either thioredoxin or glutaredoxin (硫氧化还原蛋白或谷氧化还原蛋白) can reduce the oxidized form of the reductase.

Oxidized thioredoxin and glutaredoxin are, in turn, reduced with electrons from NADPH in reactions catalyzed by thioredoxin reductase and glutathione reductase, respectively.

Glutaredoxin reduction requires two molecules of glutathione (GSH, 谷胱甘肽), resulting in oxidized glutathione (GS-SG)

• Ribonucleotide reductase uses a protein cofactor—thioredoxin or glutaredoxin—to provide electrons for reduction of the ribonucleotide substrate.

Ribonucleotide reductase has two classes of allosteric sites.

Activity sites influence catalytic efficiency, and specificity sites determine specificity for one or more of the four substrates.

Inhibition of DNA synthesis by thymidine or deoxyadenosine involves allosteric inhibition of ribonucleotide reductase by dTTP or dATP, respectively.

• Because deoxyribonucleotides are used only for DNA synthesis, and because one enzyme system is used for reduction of all four ribonucleotide substrates, regulation of both the activity and the specificity of ribonucleotide reductase is essential to maintain balanced pools of DNA precursors.

• Binding of nucleotides at the specificity sites modulates the activities if the enzyme toward different substrates, so as to maintain balanced rates of production of the four dNTPs. For example, binding of dTTP (with ATP bound in the activity site) activates the enzyme for reduction of GDP but decreases its ability to reduce either UDP or CDP.

Regulation of E. coli ribonucleotide reductase by reversible oligomerization

All nucleotides stimulate formation of the active tetramer from dimers.

dATP binding at the activity sites shifts the equilibrium to formation of inactive octamer (八聚体).

ATP binding shifts the equilibrium back to active tetramer (四聚体).

六、Thymidylate Synthase: A Target Enzyme for Chemotherapy

Biosynthesis of Thymine Deoxyribonucleotides

dUMP, the substrate for thymidylate synthesis, can arise either from UDP reduction and dephosphorylation or from deamination of a deoxycytidine nucleotide.

In the reaction catalyzed by thymidylate synthase (胸苷酸合成酶), 5,10-methylenetetrahydrofolate donates both a single-carbon methylene group and an electron pair to reduce that carbon to a methyl group.

• Ribonucleotides have multiple metabolic roles, while deoxyribonucleotides serve only as constituents of DNA
• The conversion of ribose to deoxyribose, and the conversion of uracil to thymidine，
• Both of these processes occur at the nucleotide level
• Both processes are of great interest mechanistically, as target sites for chemotherapy for cancer or infectious diseases and from the standpoint of regulation

Deoxyribonucleotide Biosynthesis and Metabolism

1. Salvage and de novo synthetic pathways to thymine nucleotides

The de novo pathways start with UDP or CDP, shown at the top.

Dashed arrows indicate feedback regulatory loops.

• A goal of chemotherapy—the treatment of diseases with chemical agents—is to exploit a biochemistry difference between the diseased tissue and the host tissue in order to interfere selectively with disease process.
• Thymidylate synthase (TS): critical for the synthesis of a deoxyribonucleotide. Any disease that involves uncontrolled cell proliferation can in principle be treated with inhibitors of TS.

Inhibition of thymidylate synthase is an approach to cancer chemotherapy, by causing specific inhibition of DNA synthesis.

• Inhibition of thymidylate synthase (胸苷酸合成酶) is an approach to cancer chemotherapy, by causing specific inhibition of DNA synthesis.

Thymidylate Synthase: A Target Enzyme for Chemotherapy

• Folate cofactor analogs (antifolates (抗叶酸药)) that compete quite effectively with 5,10-methylene-THF for binding to TS (Ki values as low as 0.4 nM).
• 5,10-Methylenetetrahydrofolate(THF)：5,10-亚甲基四氢叶酸

七、Biological and Medical Importance of Other Nucleotide Analogs

One of the earliest antiviral drugs approved for use in humans is arabinosyladenine (阿拉伯糖腺嘌呤) (araA or Vidarabine (阿糖腺苷)).

It is now being used to treat several viral diseases, including viral encephalitis (病毒性脑炎), a neurological disease caused by another member of the herpes virus family.

Unlike acycloguanosine (无环鸟苷), araA is phosphorylated to the triphosphate level by cellular kinases.

The triphosphate, araATP, is a selective inhibitor of DNA polymerases encoded by herpes viruses.

Other analogs receiving considerable attention are those being used to combat acquired immune deficiency syndrome (AIDS) caused by HIV.

One such analog, 3’-azido-2’,3’-dideoxythymidine (AZT) is anabolized to the corresponding triphosphate, which is an inhibitor of viral reverse transcriptase (the enzyme that makes a DNA copy of the viral RNA).

Other nucleoside analogs— 2’,3’-dideoxycytidine (ddC), 2’,3’-dideoxyinosine (ddI), 3’-thiacytidine (3TC), and 2’,3’-didehydro-3’-deoxythymidine (d4T)—act by conversion to the corresponding triphosphate, which is incorporated into DNA but then blocks further replicative chain elongation because of the absence of a 3’-hydroxyl terminus (chain terminators).

• 2‘，3’-双脱氧胞苷（DDC），2‘，3’-双脱氧肌苷（DDI），3‘-硫杂胞苷（3TC），和2’，3‘-双脱氢-3’-脱氧胸苷（d4T）

An important biochemical anomaly is found in parasitic protozoans (原生动物) such as Plasmodium, and Leishmania (疟原虫 利什曼原虫).

• Parasitic protozoans lack the capacity for de novo purine synthesis, and they depend entirely on salvage of nucleosides and bases provided by the host.

Compounds such as allopurinol and formycin B inhibit the growth of these organisms in culture, partly through inhibition of salvage enzymes and partly through the ability of the salvage enzymes to anabolize the analog, an ability lacking in the corresponding host enzymes.

For example, allopurinol is converted to an analog of inosinic acid (肌苷酸) and then to an AMP analog and is finally incorporated into RNA, where it interferes with messenger RNA coding in protein synthesis.

Purine salvage can also be taken advantage of, for example in the use of thiopurines (硫嘌呤)as anti-cancer and immunosuppressive drugs.

Like 5-fluorouracil, 6-mercaptopurine and 6-thioguanine (5-氟尿嘧啶，6-巯基嘌呤和6-硫鸟嘌呤) are prodrugs that require metabolic activation in order to exert their cytotoxic effects.

Another class of dihydrofolate reductase inhibitors is exemplified by trimethoprim (甲氧苄氨嘧啶).

This compound is a specific inhibitor of dihydrofolate reductases of prokaryotic origin.

Trimethoprim and its relatives are widely used to treat both bacterial infections and certain forms of malaria.

The success of these drugs derives from their being extremely weak inhibitors of vertebrate dihydrofolate reductases.

Trimethoprim is often administered in conjunction with a sulfonamide (磺胺) drug to inhibit the synthesis of folate and hence to block sequential steps in the same pathway.