Purine and Pyrimidine Metabolism Flashcards
Which bases are purines?
Adenine and Guanine
Which bases are pyrimidines?
Cytosine, Thymine and Uracil
What are the functions of nucleotide bases?
-Building blocks of nucleic acids (DNA/RNA) Involved in energy storage, muscle contraction, active transport, maintenance of ion gradients - Components of coenzymes (NAD+, NADP+, FAD, FMN, and CoA) - Activated intermediates in biosynthesis (e.g. UDP-glucose, S-adenosylmethionine) - Metabolic regulators: cAMP, cGMP, Adenylation, Uridylylation of enzyme
What is a nucleoSIDE (vs nucleoTIDE)?
A pentose sugar and the base.
What is a nucleoTIDE (vs nucleoSIDE)?
A pentose sugar, the base, and the phosphate group
What are the building blocks of the de novo synthesis pathway?
Amino acids, ribose-5-phosphate, CO2, one-carbon donors
What is the significance of inosine mono-phosphate (IMP)?
It is the first purine derivative formed in de novo synthesis. After this, there is differentiation between the AMP pathway and the GMP pathway.
Why should I know the word hypoxanthine?
Hypoxanthine is the purine base in de novo synthesis. That’s all you need to know about it.
_____ is needed for de novo purine synthesis (and TMP!); everything else is available in abundance.
folate
In de novo purine synthesis, where do the nitrogen groups come from?
Amino acids aspartate, glycine and glutamine.
In de novo purine synthesis, where do the carbons come from?
Universal carbon donor Tetrahydrofolate (abbreviated THF or FH4). This is a great single carbon donor. It’s special.
What is the most important step of de novo purine synthesis?
PRPP + Glutamine to 5’-phosphoribosylamine + Glutamate
The rest of the reactions that lead to IMP are not important now.
What is the pathway from IMP to AMP?
IMP + GTP + Aspartate = Adenylsuccinate
Adenylsuccinate + Fumarate = AMP
What is the pathway from IMP to GMP?
IMP + NAD+ + H2O = Xanthosine monophosphate (XMP)
XMP + ATP + Glutamine = GMP
How is the differentiation between creating AMP and creating GMP regulated?
AMP requires GTP
GMP requires ATP
How is purine biosynthesis regulated?
In general, product inhibition.
High AMP levels, inhibit adenylosuccinate formation
High GMP levels, inhibit XMP formation
Both high AMP & GMP levels, inhibit formation of 5-phosphoribosylamine (requires both to be high)
What are the take-home points of de novo purine synthesis?
- Precursors are amino acids (glutamine is prevalent) & folic acid (TH4 derivatives)
- Common pathway until IMP production
- Branch points to generate GMP or AMP are regulated by ATP and GTP to keep the products in balance
In pyrimidine de novo synthesis, when is PRPP added?
AFTER the pyrimidine ring has been built.
(Different from purine synthesis, where it is added in the beginning)
What is special about UMP Synthase (UMPS)?
It is bifunctional!
Orotate –> transferase –> OMP –> decarboxylase –> UMP
CPS II is used in the _____ for ______, whereas CPS I is used in the _____ for _______.
CPS II is used in the cytosol for pyrimidine biosynthesis, whereas CPS I is used in the mitochondria for urea production.
(She mentioned this like 4 times in the video.)
What is the key difference between uracil and cytosine?
Cytosine has an amino group, uracil does not.
In de novo pyrimidine synthesis, what enzyme modifies UTP to CTP? What else is needed for this reaction?
CTP Synthase is the enzyme.
The reaction also requires ATP and Glutamine
Describe Orotic Aciduria (O.A.)
- Deficiency in UMP synthase (bifunctional enzyme)
- Patients depend on diet for pyrimidines
- Excrete grams of orotic acid per day
- Bladder stones
- Mild mental retardation
- Megaloblastic anemia (results from inhibition of DNA synthesis during red blood cell production in the bone marrow)
What are the take-home points of de novo pyrimidine biosynthesis?
- First step is to make UTP ( via UMP) from basic precursors, amino acids, CO2 etc. – Sugar is added after ring is made
- CPS-II – enzyme required for carbamoyl phosphate (CP) synthesis in pyrimidine metabolism is NOT the same enzyme as CPS-I which makes CP for urea cycle
- Once UTP is made – it can be converted to CTP via NH2 transfer from glutamine at the expense of ATP
- UTP + CTP are used for RNA synthesis
- Clinical relevance : Deficiency in UMP synthase leads to orotic aciduria
In thymine synthesis, what is the key enzyme? What does it do? What doesn’t it do (or rather, what pathway option is not followed)?
The key enzyme is thymidylate synthase.
It converts dUMP to dTMP.
There is no pathway (that we are learning now) that converts dUTP to dTTP.
What are the key enzymes in thymine nucleotide synthesis?
Thymidylate synthase (dUMP –> dTMP)
Dihydrofolate reductase {DHFR} (DHFR reduces dihydrofolate to tetrahydrofolate – which can then regenerate N5,N10 methylene THF for additional reactions by TS)
How is 5-FdUMP (5-Fluorouracil) used in cancer treatment?
5-FdUMP inhibits the action of Thymidylate synthase, which in turn inhibits cell reproduction
How is methotrexate (MTX) used in cancer treatment?
Methotrexate inhibits DHFR, a key enzyme in the regeneration of N5,N10 methylene-THF, thus inhibiting the action of Thymidylate synthase.
(MTX also interferes with de novo purine biosynthesis.)
Of MTX and FdUMP, which is enhanced by folic acid supplementation?
The efficacy of FdUMP is increased with folic acid supplementation.
The efficacy of MTX is reduced with folic acid supplementation.
What is the significance of herpes viral infections with thymine nucleotide synthesis?
Herpes virus has its own TK, herpes nucleoside kinase, which works on purines and pyrimidines. This herpes TK can be targeted to prevent herpes from reproducing, while leaving the host cell (you) alone.
Is TK present in non-dividing cells?
Nope.
What are the take-home points for thymidine synthesis?
- Thymidine is required for DNA synthesis
- dTMP is created from dUMP
- dUMP to dTMP reaction is blocked by anticancer drugs FdUMP (at the level of TS enzyme reaction) and MTX (at the level of DHFR enzyme)
- Antiviral nucleoside analogs are used to treat herpes viral infections because herpes virus has its own TK – different from human TK
How do you get from RNA to DNA? (overview)
Ribonucleoside diphosphate + thioredoxin (2SH) –> *Ribonucleotide reductase* –> Deoxyribonucleoside diphosphate + thioredoxin (S-S)
How do you regenerate thioredoxin from the oxidized (S-S) state to the reduced (2 SH) state?
Thioredoxin reductase and some good ol’ NADPH
How is the pool of nucleotide bases kept in balance?
By regulating ribonucleotide reductase at two different regulation sites:
Regulation of overall activity
- ATP activates
- dATP inactivates
Regulation of substrate specificity
•ATP, dATP, dGTP, dTTP are the allosteric effector molecules
What are the take-home points of the formation of 5’-deoxyribose for DNA synthesis?
- Deoxyribonucleotides are synthesized from ribonucleotides from ribonucleotide reductase reaction
- Ribonucleotide reductase uses reduced thioredoxin to generate deoxyribonucleotides
- Reduced thioredoxin is regenerated by action of thioredoxin reductase
- Overall activity of ribonucleotide reductase is controlled by levels of dATP (inactivates) and ATP (activates) to keep a balanced pool of dNTPs
What is the overview of nitrogen excretion?
Amino Acids and Pyrimidines degrade to Urea
Purines degrade to Uric Acid
What does adenosine deaminase do?
It deaminates adenosine in the degredation of purine nucleotides, resulting in inosine.
What is xanthine oxidase?
Xanthine oxidase is a bifunctional enzyme involved in the last two steps of purine degredation. Produces uric acid.
What are the causes of gout?
Symptoms are from deposition of sodium urate crystals.
Elevated blood uric acid
In some cases from overproduction of uric acid, but in most cases from defects in renal excretion.
Diet rich in purines/alcohol
Increased purine degradation (increased cell degradation when on chemotherapy for example)
Decreased HGPRT activity (e.g. Lesh-Nyhan)
What are some treatments of gout?
Colchicine – reduces inflammation
Allopurinol – inhibits uric acid synthesis (inhibitor of xanthine oxidase enzyme)
Low purine diet - Foods that are high in purine include:
- Red meat and organ meats (eg. liver)
- Yeasts and yeast extracts (eg. beer and alcohol)
- Asparagus, spinach, beans, peas, lentils, oatmeal, cauliflower and mushrooms
Avoid caffeine and alcohol
Keep hydrated (to dilute concentration of uric acid)
What does allopurinol do for gout?
Allopurinol inhibits xanthine oxidase and lowers uric acid synthesis while increasing excretion of xanthine and hypoxanthine. Doesn’t completely inhibit, just keeps the concentration low enough to prevent crystalization.
What causes SCID? (boy in the bubble disease)
Adenine deaminase (ADA) normally deaminates deoxyadenosine as well as adenosine
ADA deficiency leads to 100X increase in dATP levels and turns off ribonucleotide reductase in both B and T lymphocytes
This inhibits proliferation in response to antigen, leading to SCID (Severe Combined Immunodeficiency)
What is going on in Lesch-Nyhan Syndrome?
Lesch-Nyhan Syndrome is due to a deficiency of Hypoxanthine-guanine phosphoribosyltransferase (HPRT), which converts hypoxanthine to IMP and guanine to GMP.
Symptoms include high uric acid – gout, mental retardation (brain cannot do de novo purine biosynthesis), cerebral palsy, self mutilation (chew on hands, etc)
What are the take-home points of Salvage Pathways for Purine Synthesis?
Purine bases created by degradation of RNA and DNA and intermediate of purines synthesis can be directly converted to the corresponding nucleotides.
The significance of the salvage pathway
1- Save energy by recycling bases
2- Breaks down excess nucleotides (from diet or tissue breakdown)
3- Some tissues and organs such as brain and bone marrow are only capable of synthesizing nucleotides by salvage pathways
Adenosine deaminase deficiency – well known cause of SCID – makes sense b/c bone marrow cannot do de novo purine synthesis
