Biochem Lecture 5: Nucleotide Metabolism

Nucleotides are made of what 3 components?

1) Nitrogenous Bases

2) Pentose Sugar

3) Phosphate Groups (1, 2, or 3)

Purine Nitrogen Bases

-two rings

Adenine

Guanine

Pyrimidine Nitrogen Bases

-one rings

Cytosine

Thymine (DNA)

Uracil (RNA)

Nucleoside

nitrogen base (adenosine, guanosine, cytidine, thymidine, and uridine), and sugar

Nucleotide

nitrogen base, sugar, and phosphate groups

Nucleotides are..?

the phosphorylation of nucleosides

What is Adenosine monophosphate an example of?

a nucleotide that has an adenine base added and one phosphate group

Deoxyribose Ring

only has 1 OH at position 3’ compared to 2 OHs in RNA at positions 2’ and 3’

-nucleotides expressed as dNMP, dNDP etc,,,

2 Paths of Nucleotide Synthesis

1) de novo synthesis

2) salvage pathways

De Novo Synthesis

the nitrogenous base itself is synthesized from simple starting materials including AAs

Salvage Pathway

preformed nitrogenous bases are recovered and attached to an activated ribose PRPP

Synthesis of Purine Nucleotides

purine ring constructed by series of reactions adding C and N to preformed ribose 5-phosphate

Where do the C and N add from?

1) aspartate adds N

2) N10- formyl- FH4 adds 2C

3) Glutamine adds 2 amide N

4) N and C from Glycine

5) CO2 adds a C

FH4

reduced form of THF, tetrahydrofolate

Synthesis of Purine Nucleotides Steps

1) starts with ribose 5-phosphate and makes PRPP, 5-phosphoribosyl-1-pyrophosphate (energy storage), using PRPP synthetase enzyme to activate ribose

2) synthesis of 5-phosphoribosylamine from PRPP

3) synthesis of inosine monophosphate using ATP energy and 2 N10-formul-THF (contributes C) and purine (adenine or guanine) attaches

PRPP synthetase Inhibition

negative feedback regulation using purine nucleotides

Azathioprine

inhibit glutamine phosphoribosyl amidotransferase enzyme preventing formation of 5-phosphorybosylamine

-immuno-suppressive and cancer drug

Mycophenolic Acid

-reversible, uncompetitive inhibitor of inosine monophosphate dehydrogenase

-drug deprives proliferating T and B cells of nucleic acids during DNA synthesis, so it can be used to prevent graft rejection

T and B lymphocyte Pathways

highly dependent on de novo pathways for proliferation

Formation of Formyl THF in Purine Synthesis

1) folate, NADPH, and dihydrofolate reductase make dihydrofolate

2) dihydrofolate, NADPH, and dihydrofolate reductase make tetrahydrofolate (THF, FH4)

3) tetrahydrofolate makes formyl-THF which contributes to the C in purine synthesis

Methotrexate

-target enzyme preventing DNA and nucleotide synthesis of cancer cells

-inhibits dihydrofolate reductase enzyme

Ribonucleotide reductase

1) makes ADP, GDP, CDP, UDP products into dADP, dGDp etc, further processing makes it into dATP, dGTP etc

2) regulates overall activity, dATP is the inhibitor of ribonucleotide reductase (so are gemcitabine, clofarabine, and cladribine)

Salvage Pathway for Purines

-most common (90%), turnover of cellular and dietary nucleic acids

Examples of Salvage Pathways for purines

Hypoxanthine uses Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and active PRPP to make IMP purine

Guanine uses Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and active PRPP to make GMP purine

Lesch-Nyhan Syndrome

-deficiency of HGPRT

-results in increased PRPP and decreased IMP and GMP nucleotides, increased de novo purine synthesis

-increased purine bases and more purine breakdown resulting in increased uric acids

-excessive production of uric acid as purines degraded (hyperuricemia)

3 Sources of Purine Nucleotides

1) Dietary purine

2) Tissue nucleic acids

3) endogenous purine synthesis

2 Important Enzymes of Purine Synthesis

1) Adenosine Deaminase (remove amino group)

2) Xanthine Oxidase (conversion to uric acid end product)

Adenosine Deaminase (ADA) deficiency

severe combined immunodeficiency (SCID)

Adenosine Deaminase to Uric Acid

-conversion of adenosine to inosine using adenosine deaminase enzyme and H2O, eventually becomes uric acid

Adenosine Deaminase in DNA

deoxyadenosine converted to deoxyinosine using adenosine deaminase enzyme and H2O, eventually becomes uric acid

Adenosine Deaminase Deficiency in DNA

-deoxyadenosine increases

-dATP (phosphorylation inhibitor) increases to inhibit ribonucleotide reductase enzyme

-this inhibits DNA synthesis and increases T and B cell apoptosis

-results in severe combined immunodeficiency (SCID)

Severe Combine Immunodeficiency (SCID) Treatments

1) bone marrow or stem cell transplants

2) injection of ADA enzyme to produce deoxyinosine

3) gene therapy

Urate

-uric acid to urate (salt conjugate base) resulting in gout

Gout

-high levels of uric acid in blood due to underexcretion or overproduction of uric acid

-deposition of sodium urate in kidneys and joints causing pain and inflammation

Gout Pathway

increase in PRPP leads to increase in purine nucleotide synthesis leading to more uric acid

3 Causes of Gout

1) abnormal PRPP synthetase activity- higher Vmax and lower Km for ribose 5-phosphate resistant to feedback regulation by nucleotides

2) defect in purine salvage pathway- increased PRPP and enhanced purine synthesis (Lesch- Nyhan syndrome)

3) alcohol

3 Treatments of Gout

1) prevention of deposition of urate crystals

2) inhibition of inflammation using colchine to decrease movement of granulocytes in deposition areas

3) inhibition of uric acid production using allopurinol, an inhibitor of xanthine oxidase

Pyrimidine C and N origins

1) has N and 3C from aspartate
2) glutamine contributes 1 amide N

3) Co2 contributes 1 C

De novo pyridine synthesis

-base is synthesized first, then attached to 5-phosphoribosyl-1-pyrophosphate (PRPP)

Orotate and De novo Pyrimidine Synthesis

carbamoyl phosphate synthetase II enzyme makes carbamoyl phosphate (first step in ammonia cycle) which combines with aspartate to make orotate (first base synthesized in pyrimidine synthesis)

CAD and Steps

trifunctional enzyme:

1) Carbamoyl phosphate synthetase II to make carbamoyl phosphate

2) Carbamoyl Phosphate and Aspartate transcarbamoylase to make carbamoylaspartate

3) carbamoylaspartate and Dihydroorotase enzyme makes dihydroorotate

4) dihydroorotate to orotate

UMP synthase

1) orotate and 5-phosphoribosyl-1-pyrophosphate (PRPP) combined with orotate phosphoribosyltransferase and urine monophosphate synthase to make ortidylate

2) orotidylate and orotidylate decarboxylase enzyme and uridine monophosphate synthase enzyme to make uridylate

Uridine Monophosphate (UMP)

first nucleotide in de novo pyrimidine synthesis

Salvage Pathways for Pyrimidine Synthesis

-use preformed pyrimidine bases recovered from nucleic acid breakdown to synthesize nucleotides

-thymine, product of DNA degradation, salvaged by first being incorporated in nucleoside by thymidine phosphorylase

Thymidine phosphorylase

thymine, deoxyribose-1-phosphate and thymidine phosphorylase make thymidine

Thymidine Kinase

thymidine, ATP, and thymidine kinase make TMP and ADP

dTMP Synthesis in Pyrimidine Synthesis

dUMP, thymidylate synthase, and N5, N10-methylene-FH4 make dTMP (dihydrofolate) which can be combines with dihydrofolate reductase to make THF and synthesize N10-methylene-FH4 again

Thymidylate Synthase Inhibition

5-fluorouracil (suicide inhibitor) produces 5-FdUMP inhibit thymidylate synthase enzyme in pyrimidine synthesis

Dihydrofolate Reductase Inhibition

methotrexate inhibits dihydrofolate reductase in pyrimidine synthesis

Ribonucleotide Reductase Inhibition

hydroxyurea inhibits ribonucleotide reductase in pyrimidine synthesis, RR produces dUDP which turns to dUMP

Tetrahydrofolate

one carbon metabolism present in purine (N10-formyl-FH4) and pyrimidine (N5, N10-methylene-FH4) synthesis

THF (FH4) in AA Metabolism

1) degradation of histidine

2) serine synthesis

3) resynthesis of methionine from homocysteine

THF Primary 1C Carrier, 1C Pool

carry and transfer oxidation state of one carbon unit (methane, methanol, formic acid)

Sources of 1C Units

serine, glycine, histidine, formaldehyde, formate

Folate

nutrients come directly from food sources, vitamin B9

-half of the body’s folate storage in fully reduce form THF or FH4 in liver and conjugated with 7 glutamates

Folic Acid

synthetic vitamin B9 manufactured for use as supplement to fortify foods

-humans can’t synthesize folic acids and need external source for this vitamin

Sulfonamides

structural analogs of PABA (para-aminobenzoic acid) that competitively inhibit bacterial synthesis of folic acid bc purine synthesis needs THF as coenzyme the drugs slow pathway in bacteria

-no effect in human purine synthesis

Tetrahydrofolate Derivatives

1) N10-formyl-FH4 most oxidized form make purines

2) N5, N10- methenol-FH4 enters serine ( 1C source) combines with glycine make thymine

3) N5-methyl-FH4 can’t be reoxidized and is the most reduced form and make methionine

Methyl Folate Trap

deficiency of vitamin B12

-reaction converting homocysteine to methionine can’t proceed and 5-methyl-THF accumulates and folate becomes trapped

-decrease in THF, decrease in dTMP, purine, and DNA synthesis