Nucleotide+Metabolism

=**Objectives**=


 * 1. Identify the structure of the five common nitrogenous bases found in nucleotides that are incorporated into nucleic acids. Name the nucleosides and nucleotides derived from these bases** (p. 8)


 * **Base** || **Ribose Nucleoside** || **Deoxyribose Nucleoside** ||
 * [|Adenine] || Adenosine || Deoxyadenosine ||
 * [|Guanine] || Guanosine || Deoxyguanosine ||
 * [|Thymine] || -- || (Deoxy) Thymidine ||
 * [|Uracil] || Uridine || -- ||
 * [|Cytosine] || Cytidine || Deoxycytidine ||

Nucleotides are named after the nucleoside, **not** the base, i.e. adenosine monophosphate rather than adenine monophosphate.


 * 2. Identify the nitrogenous base, sugar, phosphate, glycosidic bond, phosphomonoester bond, and phosphoanhydride bonds of a nucleoside triphosphate** (p. 7)

Nitrogenous base is the purine or pyrimidine. Sugar is the attached ribose/deoxyribose sugar. Glycosidicbond is the bond between the nitrogenous base and the sugar. Phosphomonoester bond is the bond between the sugar and the phosphate. Phosphoanhydride bonds are the bonds between adjacent phosphate groups.


 * 3. Name the committed steps in the //de novo// purine and pyrimidine biosyntheic pathways.** (p. 10)

__Purine Synthesis__ PRPP + Glutamine + H2O --> Phosphoribosylamine + Glutamate + PPi, catalyzed by PRPP Glutamyl Aminotransferase. Glutamine acts as an amine donor, committing ribose-5P to purine synthesis

__Pyrimidine Synthesis__ CO2 + Glutamine + ATP --> carbamoyl phosphate (CAP), catalyzed by carbamoyl phosephate synthetase II. Formation of carbamoyl does not necessary commit to the synthesis of pyrimidines but, in humans, carbamoyl phosphate synthetase II, aspartate transcarbamoylase, and dihydrorotase are combined as one protein called CAD. Formation of carbamoyl by CAD channels it through the next two steps of pyrimidine synthesis so it is considered the "committed step."

Carbamoyl phosphate synthetase I is found in mitochondria in the urea cycle and uses ammonia as its amine source. Carbamoyl phosphate synthetase II is found in the cytosol in pyrimidine synthesis and uses glutamine as its amine source.


 * 4. Compare and contrast the biosynthetic pathways for purines and pyrimidines.** (p. 11)

__Purine Synthesis__ Purine ring is built onto ribose-5P. Ring atoms are added one at a tie from simple intermediates, except for 3 ring carbons from glycine. Glutamine serves as an amino group donor in steps 2, 5, and 15. Formyl tetrahydrofolate serves as carbon donor in steps 4 and 10. The first nucleotide formed is IMP, which can be converted to AMP or GMP by a branched pathway.

__Pyrimidine Synthesis__ Pyrimidines are made from simple abundant cellular compounds: CO2, aspartate, and amide from glutamine All ring atoms are present after the second step Ribose is added after the ring is formed Source of ribose is PRPP, just like in purine synthesis Three major end products are UTP, CTP, and TMP


 * 5. Identify the principle metabolic source of one-carbon units that are metabolized via tetrahydrofolate.**

The -CH3 unit used by tetrahydrofolate for single-carbon donation comes from a serine. Serine + H4-folate --> Glycine + 5,10-methylene H4 folate + H2O


 * 6. State the first purine nucleotide that is formed during //de novo// purine synthesis.** (p. 10-11)

Inosine monophosphate (IMP), which can then be converted to AMP or GMP.


 * 7. State the first pyrimidine nucleotide that is formed during //de novo// pyrimidine synthesis.**

Orotate monophosphate (OMP), which can be converted to UMP and further modification to CTP and TMP


 * 8. Name the two steps that are regulated early in the //de novo// purine biosynthetic pathway.** (p. 12)

(1) Ribose-5P + ATP --> Phosphoribosyl-pyrophosphate (PRPP), catalyzed by PRPP Synthetase (2) PRPP + Glutamine + H2O --> Phosphoribosylamine + Glutamate + PPi, catalyzed by PRPP Glutamyl Aminotransferase.

Both steps are inhibited by negative feedback of IMP, AMP, and GMP.


 * 9. Name the enzyme that is regulated early in the //de novo// pyrimidine biosynthetic pathway of mammals.**

Carbamoyl phosphate synthetase II is inhibited by negative feedback from UTP.


 * 10. State the enzyme of nucleotide metabolism that is inhibited by a given antimetabolite.** (p. 14)

Antimetabolites are synthetic analogs of enzyme substrates/cofactors that block metabolite pathways by inhibiting enzymes.

__Glutamine Analogs__ Azaserine -- inhibit aminotransferases in purine synthesis (steps 2 and 5); prevent amine group donation. Additionally, azaserine can inhibit conveson of UTP to CTP in pyrimidine synthesis, a reaction requiring glutamine as an amino donor/

__Purine Nucleotide Analogs__ Mercaptopurine -- After conversion to ribonucleotides, purine nucleotide analogs block purine synthesis by acting as feedback inhibitors of PRPP amino-transferase, the first commited step of purine synthesis.

These drugs have a long history for treatment of acute pediatic leukemia.

__Pyrimidine Nucleotide Analogs__ 5-flourouracil -- inhibits thymidylate synthesis. 5-flourouracil is converted to fdUMP (5-fluoro-2'-deoxyuridine-5'-phosphate) by enzymes in the salvage pathway and binds irriversibly to the active site of thymidylate synthase.

5-flourouracil is used for teh treatmet of solid tumors.

__Antifolates__ Methotrexate, aminopterin, and other folate analogs inhibit dihydrofolate reductase (DHFR). This prevents the regeneration of the active form of folate, formyl tetrahydrofolate, which is necessary for step 4 and 10 in purine synthesis.

Sulfonamide Drugs -- analog of p-aminobenzoic acid, inhibiting folate synthesis in bacteria.


 * 11. State the level of phosphorylation at which ribonucleotides are converted to deoxyribonucleotides.**

Ribonucleotides need to be in the Diphosphorylated state for conversion to deoxyribonucleotides by ribonucleotide reductase.

After conversion to deoxyribonucleotides, thymine deoxyribonucleotides must be made from deoxyuridine //mono//phosphate by thymidylate synthase. However, conversion of ribonucleotides to deoxyribonucleotides still requires the diphosphate level.


 * 12. State which nucleotides allosterically inhibit ribonucleotide reductase, and why high levels of thymidine arrest growth of cells in culture**

Ribonucleotide reductase is inhibited generally by dATP at the activity determining site. dGTP and dTTP act as inhibitors on the specificity-determining site. Specifically, dTTP inhibits formation if dUDP, but promotes formation of dGDP and dGTP inhibits formation of dGTP, but promotes formation of dADP.

High levels of thymidine can block cell growth in culture by blocking DNA synthesis. Adding thymine increases the pools of dTTP, dGTP, and dATP but decreases the pool of dCTP. This is because dTTP promotes synthesis of dGTP and dADP by ribonucleotide reductase while dTTP, dGTP and dADP all inhibit synthesis of dCTP. Reduction of UDP to dUDP for conversion to dTTP is also inhibited but dTTP can be made directly from thymidine, independent of ribonucleotide reductase. This deficiency in dCTP prevents DNA synthesis and blocks cell growth.


 * 13. Describe the thymidylate synthase reaction and its role in nucleotide synthesis.**

Deoxyribonucleotides are formed by reduction of already existing ribonucleotides, forming ADP, GDP, CDP, and UTP. However, thymine rather than uracil is found in DNA so thymine deoxyribonucleotides must be synthesized.

dUMP + 5,10-methylene H4 folate --> dTMP + dihydrofolate, catalyzed by thymidylate synthase.


 * 14. Diagram the two "preformed" or "salvage" pathways of nucleotide synthesis.**

(1) PRPP + Purine --> Purine ribonucleotide + PPi, catalyzed by phosphoribosyl transferase.

Specificity: Adeninephosphoribosyl Transferase (APRT) Hypoxanthine-Guanine Phosphoribosyl Transfers (HGPRT) Uracil Phosphoribosyl Transferse (UPRT)

Single step from free base to nucleotide. Cytosine does not participate in this reaction. No deoxy compound analogous to PRPP exists to make deoxynucleotides directly.

(2a) Thymine + Deoxyribose-1P <--> Thymidine = Pi, catalyzed by Nucleoside phosphorylases (2b) Thymidine + ATP --> dTMP + ADP, catalyzed by Nucleoside kinase

Two step process. Functions only for uracil (with either deoxyribose-1P or ribose-1P) and thymine (only with deoxyribose-1P). Does not function with adenine, guanine, or hypoxanthine.


 * 15. State the end product of purine catabolism in humans and the precursors that xanthine oxidase acts upon to yield this end product.**

The end product of purine catabolism in humans is uric acid. xanthine oxidase oxidzes hypoxantine to xanthine; xanthine oxidase then oxidizes xanthine again to produce uric acid.


 * 16. State the end products of pyrimidine catabolims in humans.**

The main end products of pyrimidine catabolism in humans are CO2 and NH3. Beta-aminoisobutyrate is also formed exclusively from the degredation of thymine and excreted in the urine. Beta-aminoisobutyrate can be used to estimate the DNA turnover rate in cancer patients.


 * 17. State the known enzymatic defects in patients with Gout, Lesch-Nyhan Syndrome, Orotic aciduria, Xanthinuria, and Severe Combined Immunodeficiency. Describe treatments for these diseases, when available, and their mechanisms of action.** (p. 13-14)

__Gout__ Inflammation of joints due to sodium urate crystal deposits, often in the big toe, leading to acute and painful gout attacks. Related to hyperuricemia -- excess uric acid in circulation and urine.

Defects in purine metaboism may predispose to gout: (1) PRPP Synthase deficiency -- Mutant form is not regulated by phosphate or nucleotides (2) Glucose-6-phosphatase deficiency -- Increased glucose-6P leads to increased ribose-5P from the pentose phosphate cycle. (3) Partial HGPRTase deficiency -- an enzyme of the salvage pathway

Increased purine synthesis from (1) and (2), or ineffective salvaging of purines (3), can lead to increased uric acid from purine degradation. Defect in clearance of uric acid by kidneys can result in gout.

Treatment options:

Anti-inflammatory (1) Colchicine -- alkaloid, reduces inflammation of joints by inhibiting microtubule assembly in leukocytes (2) Ibuprofen -- standard non-steroid anti-inflammation drugs

Inhibit uric acid production (1) Allopurinol -- inhibits xanthine oxidase. Xanthine oxidase converts allopurinol to alloxanthine, which inhibits xanthine oxidase (suicide inhibition).

Incrase uric acid clearance (1) Prebenecid -- uricosuric drug, increases renal clearance by inhibiting reabsorbtion of uric acid.

__Lesch-Nyhan Syndrome__ Absense of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) causes Lesch-Nyhan syndrome, characterized by excessive uric acid production, and neurological problems including self-mutilation,choreoathetosis, spasticity, and mental retardation.

Lack of HGPRT prevents salvaging of guanine and hypoxanthine, which shunts the unused PRPP to //de novo// synthesis of purine nucleotides which raises levels of uric acid. Neurological problems seem to be due to the brain's reliance on the salvage pathway for nucleotides in RNA and DNA synthesis. Although purine synthesis is increased as a result of HGPRT deficiency, the brain cannot use these newly synthesized purines.

There is no effective treatment for the neurological problems associated with Lesch-Nyhan Syndrome though inhibitors of uric acid production and promotors of uric acid clearance can be used to treat the hyperuricemia.

__Orotic Aciduria__ Megaloblastic anemia, growth retardation, excess orotic acid in urine. Caused by deficinecy in orotine-5P pyrophosphorylase and orotidine-5P decarboxylase -- the two enzymes forming the protein UMP sythase in steps 5 and 6 of pyrimidine synthesis.

Deficiency in UMP synthase results in lack of pyrimidines for DNA and RNA synthesis in rapidly dividing cells. However, because this blockage also prevents synthesis of UTP, the feedback inhibition on CPSII fails, leading to increase flux through the pyrimidine synthesis pathway and accumulation of more orotic acid.

Orotic aciduria can be treated by giving uridine orally. Uridine becomes converted to UTP in the body and will help to inhibit CPSII to down regulate the pathway. Additionally, other pyrimidines (C and T) can be made from uridine nucleotides.

__Xanthinuria__ Genetic deficiency in xanthine oxidase, causing increased excretion of xanthine and hypoxanthine and decreased uric acid production. Severe cases may result in xanthine lithiasis (kidney stones).

Xanthine oxidase catalyzes the converstion of (1) hypoxanthine to xanthine catabolism of adenosine and (2) conversion of xanthine to uric acid in purine catabolism.

(1) Hypoxanthine + H2O + O2 --> Xanthine + H2O2 (2) Xanthine + H2O + O2 --> Uric acid + H2O2

__Severe Combined Immunodeficiency__ Deficiency in adenosine deaminase can cause SCID (no T or B cells of immune system). May be caused by buildup of dATP which inhibits ribonucleotide reductase, the first step in adenosine catabolism. This prevents DNA synthesis and subsequent B and T cell development. Some treatment trials with gene therapy.

Deficiency in purine nucleoside phosphorylase can induced deficiency in T cells, but normal B cells. May be caused by buildup of dGTP, which inhibits reduction of pyrimidine ribonucleotides by ribonucleotide reductase, and, hence, DNA synthesis.