Purine and Pyrimidine Metabolism

Question 1

Which bases are purines?

Answer 1

Adenine and Guanine

Question 2

Which bases are pyrimidines?

Answer 2

Cytosine, Thymine and Uracil

Question 3

What are the functions of nucleotide bases?

Answer 3

-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

Question 4

What is a nucleoSIDE (vs nucleoTIDE)?

Answer 4

A pentose sugar and the base.

Question 5

What is a nucleoTIDE (vs nucleoSIDE)?

Answer 5

A pentose sugar, the base, and the phosphate group

Question 6

What are the building blocks of the de novo synthesis pathway?

Answer 6

Amino acids, ribose-5-phosphate, CO2, one-carbon donors

Question 7

What is the significance of inosine mono-phosphate (IMP)?

Answer 7

It is the first purine derivative formed in de novo synthesis. After this, there is differentiation between the AMP pathway and the GMP pathway.

Question 8

Why should I know the word hypoxanthine?

Answer 8

Hypoxanthine is the purine base in de novo synthesis. That’s all you need to know about it.

Question 9

_____ is needed for de novo purine synthesis (and TMP!); everything else is available in abundance.

Answer 9

folate

Question 10

In de novo purine synthesis, where do the nitrogen groups come from?

Answer 10

Amino acids aspartate, glycine and glutamine.

Question 11

In de novo purine synthesis, where do the carbons come from?

Answer 11

Universal carbon donor Tetrahydrofolate (abbreviated THF or FH4). This is a great single carbon donor. It’s special.

Question 12

What is the most important step of de novo purine synthesis?

Answer 12

PRPP + Glutamine to 5’-phosphoribosylamine + Glutamate

The rest of the reactions that lead to IMP are not important now.

Question 13

What is the pathway from IMP to AMP?

Answer 13

IMP + GTP + Aspartate = Adenylsuccinate

Adenylsuccinate + Fumarate = AMP

Question 14

What is the pathway from IMP to GMP?

Answer 14

IMP + NAD+ + H2O = Xanthosine monophosphate (XMP)

XMP + ATP + Glutamine = GMP

Question 15

How is the differentiation between creating AMP and creating GMP regulated?

Answer 15

AMP requires GTP

GMP requires ATP

Question 16

How is purine biosynthesis regulated?

Answer 16

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)

Question 17

What are the take-home points of de novo purine synthesis?

Answer 17

1. Precursors are amino acids (glutamine is prevalent) & folic acid (TH4 derivatives)
2. Common pathway until IMP production
3. Branch points to generate GMP or AMP are regulated by ATP and GTP to keep the products in balance

Question 18

In pyrimidine de novo synthesis, when is PRPP added?

Answer 18

AFTER the pyrimidine ring has been built.

(Different from purine synthesis, where it is added in the beginning)

Question 19

What is special about UMP Synthase (UMPS)?

Answer 19

It is bifunctional!

Orotate –> transferase –> OMP –> decarboxylase –> UMP

Question 20

CPS II is used in the _____ for ______, whereas CPS I is used in the _____ for _______.

Answer 20

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.)

Question 21

What is the key difference between uracil and cytosine?

Answer 21

Cytosine has an amino group, uracil does not.

Question 22

In de novo pyrimidine synthesis, what enzyme modifies UTP to CTP? What else is needed for this reaction?

Answer 22

CTP Synthase is the enzyme.

The reaction also requires ATP and Glutamine

Question 23

Describe Orotic Aciduria (O.A.)

Answer 23

• 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)

Question 24

What are the take-home points of de novo pyrimidine biosynthesis?

Answer 24

1. First step is to make UTP ( via UMP) from basic precursors, amino acids, CO2 etc. – Sugar is added after ring is made
2. 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
3. Once UTP is made – it can be converted to CTP via NH2 transfer from glutamine at the expense of ATP
4. UTP + CTP are used for RNA synthesis
5. Clinical relevance : Deficiency in UMP synthase leads to orotic aciduria

Question 25

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)?

Answer 25

The key enzyme is thymidylate synthase. It converts dUMP to dT**M**P. There is no pathway (that we are learning now) that converts dUTP to dT**T**P.

Question 26

What are the key enzymes in thymine nucleotide synthesis?

Answer 26

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)

Question 27

How is 5-FdUMP (5-Fluorouracil) used in cancer treatment?

Answer 27

5-FdUMP inhibits the action of Thymidylate synthase, which in turn inhibits cell reproduction

Question 28

How is methotrexate (MTX) used in cancer treatment?

Answer 28

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.)

Question 29

Of MTX and FdUMP, which is enhanced by folic acid supplementation?

Answer 29

The efficacy of FdUMP is **increased** with folic acid supplementation. The efficacy of MTX is **reduced** with folic acid supplementation.

Question 30

What is the significance of herpes viral infections with thymine nucleotide synthesis?

Answer 30

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.

Question 31

Is TK present in non-dividing cells?

Answer 31

Nope.

Question 32

What are the take-home points for thymidine synthesis?

Answer 32

1. Thymidine is required for DNA synthesis 2. dTMP is created from dUMP 3. 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) 4. Antiviral nucleoside analogs are used to treat herpes viral infections because herpes virus has its own TK – different from human TK

Question 33

How do you get from RNA to DNA? (overview)

Answer 33

Ribonucleoside diphosphate + thioredoxin (2SH) --\> *\*Ribonucleotide reductase\** --\> Deoxyribonucleoside diphosphate + thioredoxin (S-S)

Question 34

How do you regenerate thioredoxin from the oxidized (S-S) state to the reduced (2 SH) state?

Answer 34

Thioredoxin reductase and some good ol' NADPH

Question 35

How is the pool of nucleotide bases kept in balance?

Answer 35

By regulating *ribonucleotide reductase* at two different regulation sites: ## Footnote **Regulation of overall activity** * ATP activates * dATP inactivates **Regulation of substrate specificity** •ATP, dATP, dGTP, dTTP are the allosteric effector molecules

Question 36

What are the take-home points of the formation of 5’-deoxyribose for DNA synthesis?

Answer 36

1. Deoxyribonucleotides are synthesized from ribonucleotides from ribonucleotide reductase reaction 2. Ribonucleotide reductase uses reduced thioredoxin to generate deoxyribonucleotides 3. Reduced thioredoxin is regenerated by action of thioredoxin reductase 4. Overall activity of ribonucleotide reductase is controlled by levels of dATP (inactivates) and ATP (activates) to keep a balanced pool of dNTPs

Question 37

What is the overview of nitrogen excretion?

Answer 37

Amino Acids and Pyrimidines degrade to Urea Purines degrade to Uric Acid

Question 38

What does adenosine deaminase do?

Answer 38

It deaminates adenosine in the degredation of purine nucleotides, resulting in inosine.

Question 39

What is xanthine oxidase?

Answer 39

Xanthine oxidase is a bifunctional enzyme involved in the last two steps of purine degredation. Produces uric acid.

Question 40

What are the causes of gout?

Answer 40

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)

Question 41

What are some treatments of gout?

Answer 41

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)

Question 42

What does allopurinol do for gout?

Answer 42

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.

Question 43

What causes SCID? (boy in the bubble disease)

Answer 43

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)

Question 44

What is going on in Lesch-Nyhan Syndrome?

Answer 44

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)

Question 45

What are the take-home points of Salvage Pathways for Purine Synthesis?

Answer 45

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