Nucleotide Metabolism (Purines and Pyrimidines)

Question 1

Ribonucleosides

Answer 1

• Adenine (A)- Adenosine, AMP
• Guanine (G)- Guanosine, GMP
• Uracil (U)- Uridine, UMP
• Cytosine (C)- Cytidine, CMP
• Hypoxanthine- Inosine, IMP

Question 2

Deoxyribonucleoside

Answer 2

• Adenine (A)- Deoxyadenosine, dAMP
• Guanine (G)- Deoxyguanosine, dGMP
• Uracil (C)- Deoxyuridine, dUMP
• Cytosine (C)- Deoxycytidine, dCMP
• Thymine (T)- Deoxythymidine, dTMP

Question 3

Purine bases

Answer 3

• adenine
• guanine
• hypoxanthine
• xanthine

Question 4

Pyramidine bases

Answer 4

• uracil
• cytosine
• thymine

Question 5

PRPP

Answer 5

• 5’-Phosphoribosyl-1-pyrophosphate is an intermediate of major significance in nucleotide metabolism
• formed from ribose 5-phosphate (from glucose metabolism, pentose phosphate shunt or can do nucleoside degradation) and adenosine triphosphate (ATB) catalyzed by PRPP synthetase

Question 6

PRPP required for:

Answer 6

• de novo synthesis of pyrimidine and purine nucleotides
• salvage pathways for purine nucleotides
• biosynthesis of nucleotide coenzymes, NAD and FAD

Question 7

Synthesis of purines

Answer 7

• base- the purine ring is built on a molecule of PRPP
• the precursprs of the ring are glutamine (glutamine amino transfer reactions), glycine, CO2, aspartate (aspartate to malate conversion), and two one carbon fragments from the one carbon folate pool(from THF)
• synthesis of inosine 5’-monophosphate (IMP). The formation of IMP is a ten-step process that uses six high-energy phosphate bonds. Energetically expensive

Question 8

Inhibition of IMP synthesis

Answer 8

• azaserine (analog of glutamine) blocks amide transfer from glutamine
• sulfonamides (analogs of p-aminobenzoic acid) block the biosynthesis of folic acid in bacteria and prevent its formation. This blocks nucleotide synthesis

Question 9

Important features of IMP biosynthesis

Answer 9

• the first step is the rate limiting and regulated step
• two steps require folate, and are blocked by drugs that block folate biosynthesis in bacteria
• the nucleotide ring is made from glutamine, glycine, carbon dioxide, asparate, and two one-carbon fragments from the one carbon folate pool
• two steps require glutamine amino transfer reactions that are inhibited by azaserine
• this is an expensive process and we would just as soon salvage all of our nucleotides/ 90% of our nucleotides are salvaged

Question 10

De novo synthesis of purine nucleotides is regulated by feedback inhibition

Answer 10

• the enzymes that catalyze the first two steps of IMP synthesis- PRPP synthetase and PRPP amidotransferase- are both inhibited by IMP, GMP, and AMP
• PRPP amindotransferase has two allosteric sites, one for IMP or GMP and one for AMP. IF both sites are occupied, then inhibition is synergistic
• inhibition: the synthesis of adenylosuccinate from IMP is inhibited by AMP, and the synthesis of XMP is inhibited by GMP
• substrate channeling and intracellular localization, the purinosome
• nucleoside monophosphate kinase- GMP + ATP GDP + ADP
• nucleoside diphosphate kinase- GDP + ATP GTP + ADP

Question 11

Salvage pathways for purine nucleotides

Answer 11

-ribose phosphate transfer enzymes. There are two specific enzymes that catalyze the transfer of the ribose phosphate from PRPP to free purine bases, which are formed by the degradation of nucleotides
-HGPRT- catalyzes the formation of nucleotides from either hypoxanthine or guanine, inhibited by IMP and GMP
Hypoxanthine + PRPP -> IMP + PPi
Guanine + PRPP -> GMP + PPi

-adenine phosphoribosyl transferase (APRT)- catalyzes the formation of AMP from adenine, inhibited by AMP
Adenine + PRPP -> AMP + PPi

-nucleoside kinase- only adenosine in humans and not widely used
Adenosine + ATP -> AMP + ADP

-about 90% of the free purine in the body is salvaged and recycled

Question 12

Purinosome

Answer 12

• in humans, purine biosynthetic machinery localized to the purinosome
• presence of this organelle is regulated by purine abudance
• all ten steps catalyzed by six different enzymes localize in these organelles along with the purine salvage pathway enzymes

Question 13

Synthesis of pyrimidine nucleotides

Answer 13

-base- not built on PRPP, instead pyrimidine ring is formed and then reacts with PRPP to form the nucleotide
-the precursors of the ring are carbamoylphosphate and aspartate
-the formation of uridine 5’ monophosphate- the first compound in the pathway is carbomoylphosphate (synthesized in cytosol from glutamine and carbon dioxide, in liver as an intermediate in urea synthesis, but this synthesis takes place in the mitochondria and is catalyzed by a different enzyme
(first three steps by single enzyme complex, next two by another enzyme complex- substrate channeling)
-UMP is coverted to UTP in two steps
-UTP to form CTP- glutamine amino-transferase, activated by GTP)

Question 14

Regulation of pyrimidine synthesis

Answer 14

• enzyme- a single protein that consists of three identical subunits catalyzes the first three steps of the synthesis
• the last two steps are catalyzed by another multifunctional enzyme
• pyrimidine nucleotides- decrease the aspartate transcarbamoylase and dihydroorotase activities of the multifunctional enzyme, but the molecular basis for decrease in activity is unknown

Question 15

Salvage pathways for pyrimidine nucleotides

Answer 15

• uracil can be concerted to UMP:
• catalyzed by a nucleoside phosphorylase, uridine phosphorylase: uracil + ribose 1-phosphate -> Uridine + Pi
• second reaction catalyzed by uridine kinase: uridine +ATP -> UMP + ADP
• cytidine can also be phosphorylated by uridine kinase

Question 16

Enzymes that use glutamine as a NH3 donor

Answer 16

1) CPSII- carbamoyl phosphate synthase II- produce carbamyol phosphate in the cytosol, pyrimidine biosynthesis
2) CTP Synthase-pyrimidine biosynthesis (interconverts UTP and CTP)
3) FGAR Aminotransferase
4) GMP Synthase
5) PRPP Aminotransferase- important for purine
6) Asparagine Synthase

Question 17

Deoxyribonucleotides

Answer 17

• found in DNA, ordinarily low and they increase at the time of DNA replication- 99.9% cells aren’t making, only cells in S phase
• formed by reduction of only ribonucleoside diphosphates

Question 18

Ribonucleotide reductase

Answer 18

• reduction of ribonucleoside diphosphates
• regulation- by ribo and deoxyribonucleotides in a complex fashion- ensures that proper ratios and levels of deoxyribonucleotides are present for the synthesis of DNA
• products are dADP, dCDP, dGDP, dUDP
• inhibited by hydroxyurea- chemo agent
• cells fo not want to incorporate deoxy-U into DNA, so this molecule is efficiently dephosphorylated to dUMP state

Question 19

Thymidylate synthase

Answer 19

• dTMP is formed from dUMP in a reaction catalyzed
• in this reaction, a methylene group from N5N1–methylenetetrahydrofolate is oxidized to dihydrofolate
• tetrahydrofolate is regenerated by dihydrofolate reductase in a reaction that requires NADPH
• this enzymes is also carefully regulated in a cell cycle dependent manner, and is made by cells in S- phase
• methyl group comes from conversion of Serine to Glycine which generates N5, N10 methylene THF
• serine can be easily synthesized from intermediates of glycolosis: so one carbon groups can be funneled right out of glycolosis and onto dUMP to make dTMP

Question 20

5- Fluorouracil

Answer 20

• undergoes conversion to nucleoside 5’ monophosphate F-UMP in cells
• F-UMP can be phosphorylated to the nucleoside diphosphate- reduced to deoxyribonucleotide
• F-dUMP-reduced nucleoside diphosphate is dephosphylated to 5-fluorodeoxyuridine 5’-monophosphate (F-dUMP), which is critical form for antineoplastic activity
• F-dUMP interacts with thymidylate synthase and N5N10-methylenetetrahydrofolate
• inhibition: the transfer of a methylene group to the pyrimidine ring is blocked by the fluorine atom. Therefore, thymidylate synthase is trapped in a complex with F-dUMP, and synthesis of dTMP is inhibited

Question 21

Methotrexate

Answer 21

• an analog of folic acid that inhibits dihydrofolate reductase
• the regeneration of tetrahydrofolate is blocked, and synthesis of dTMP is inhibited

Question 22

Salvage pathway for the synthesis of thymine deoxyribonucleotides

Answer 22

-thymine phosphorylase converts thymine to thymindine
Thymine + Deoxyribose 1-phosphate -> Thymidine + Pi
-thymidine kinase converts thymidine to dTMP
Thymidine +ATP -> dTMP + ADP

Question 23

Degradation of Purine and Pyrimidine Nucleotides

Answer 23

• nucleotides remove the 5’-phosphates from purine and pyrimidine ribo- and deoxyribonucleotides, converting them to ribo- and deoxyribonucleosides
• nucleoside phosphorylase catalyze the phosphorolysis of nucleosides to free bases and ribose 1-phosphate or deoxyribose 1-phosphate

Question 24

Nucleotidases

Answer 24

• remove 5’ phosphates from purine and pyrimidine ribo- and deoxyribonucleotides, converting them to ribo- and deoxyribonucleosides
• AMP is degraded by two pathways: it may first be deaminated to IMP, with IMP then hydrolyzed to inosine, or it may be hydrolyzed to adenosine first, with the adenosine then deaminated to inosine
• GMP is hydrolyzed to guanosine
• cytidine monophosphate (CMP) may be deaminated to UMP and then hydrolyzed to uridine, or it may be hydrolyzed to cytidine and then deaminated to uridine.
• UMP is hydrolyzed to uridine

Question 25

Nucleoside phosphorylases

Answer 25

-catalyze the phosphorolysis of nucleosides to free bases and ribose 1-phosphate or deoxyribose 1-phosphate -in phosphorolysis, a bond is cleaved by the addition of Pi across it: Inosine + Pi -> Hypoxanthine + Ribose 1 phosphate Guanosine + Pi -> Guanine + Ribose 1 phosphate Uridine + Pi -> Uracil + Ribose 1 phosphate Thymidine + Pi -> Thymine + Deoxyribose 1-phosphate -nucleoside phosphorylases readily catalyze the reverse reaction, the conversion of a free base to a nucleoside. The reverse reaction is important in salvage pathways, especially of uracil and thymine

Question 26

Purine Degradation

Answer 26

- the final product of purine degradation is uric acid, which is produced via the following pathway - hypoxanthine-from the breakdown of AMP, is oxidized to xanthine by the enzyme xanthine oxidase - guanine- from the breakdown of GMP, is deaminated to xanthine - xanthine is oxidized to uric acid by xanthine oxidase - oxygen (O2) is required and superoxide is formed. The O2 is converted to hydrogen peroxide by superoxide dismutase - xanthine oxidase contains molybdenum, which is why this element is reqired in trace amounts in humans. This enzyme also contains iron and sulfur. This is the only known use of molybdenum in humans

Question 27

Purine Degradation Overview

Answer 27

1) dephosphoylation 2) deamination 3) separation of the base from the ribose 4) oxidation to uric acid the first three don't necessarily happen in any specific order

Question 28

Degradation of Pyrimidines

Answer 28

- the final products of pyrimidine degradation of Beta-alanine (from uracil) and Beta-aminoisobutyrate (from thymine) - they also undergo dephosphorylation, separation of base from ribose, deamination and degredation of the base

Question 29

Beta- aminoisobutyric acid

Answer 29

-marker for cancer cell death

Question 30

Gout

Answer 30

- precipitation of sodium urate crystals in the joints and kidneys - sodium urate crystals precipitate because the serum levels of urate exceed its solubility limit - elevated uric acid levels may be due to one of several disorders - in some patients, PRPP synthetase is abnormal and is not responsive feedback inhibition by purine nucleoside diphosphates - partial deficiency of HGPRT leads to increased cellular levels of PRPP which leads to increased de novo synthesis of purines - this partial deficiency does not cause any of the neurologic symptoms of Lesch Nyhan syndrome

Question 31

Effect of Gout

Answer 31

- inflammation and erosion of the joints occur when leukocytes engulf the deposited crystals and consequently rupture, releasing lysosomal enzymes - sodium urate crystals in the urinary tract impair renal function

Question 32

Treatment of Gout

Answer 32

- Allopurinol- is an important drug in the treatment of gout: it blocks the production of uric acid. Also colchicine is used - allopurinol is oxidized by xanthine oxidase to oxypurinol - oxypurinol binds tightly to xanthine oxidase, inhibiting its ability to oxidize xanthine or hypoxanthine- suicide inhibition - often given as a pretreatment for chemo: during chemo large quantities of nuclotides are released from killed cancer cells-> uricemia and gout

Question 33

Lesch-Nyhan syndrome

Answer 33

- hereditary X-linked recessive condition due to a severe or complete deficiency of HGPRT activity - increased synthesis of purines= little HGPRT so hypoxanthine and guanine are not salvaged, also intracellular levels of PRPP increase, while those IMP and GMP decrease leads to de novo synthesis of purines - several forms of HGPRT deficency - one form normal levels but inactive, some patients have unstable so higher activity in young red blood cells - symptoms: hyperuricemia, gout, urinary tract stones,mental retardation, spasticity, and self-mutilation - treatment with allopurinol reduces the uric acid formation but does not alleviate the neurologic symptoms

Question 34

Adenosine deaminase deficiency

Answer 34

- in cells of people with ADA deficiency, deoxyadenosine and adenosine are abundant because they are not degraded to deoxyinosine and inosine - they are however efficiently converted to nucleotides in white blood cells - the high levels of dATP inhibit ribonucleotide reductase which inhibits DNA snythesis - therefore white blood cells cannot proliferate - ADA deficiency is associated with severe combined immunodeficiency (SCID) - bone marrow transplantation ans enzyme replacement have been used to treat SCID. Gene therapy is an experimental treatment