Nucleotide Metabolism (Week 4--Kurdistani) Flashcards

Question

Where do the 6 steps of pyrimidine de novo biosynthesis occur, and what is the significance of this?

Answer 1

All steps occur in cytoplasm, except step 4 which is in the **mitochondria** Step 4 is **dihydroorotate --\> orotate** (via dihydroorotate dehydrogenase, enzyme that is in the intermembrane space of the mitochondria) Significance is that this links synthesis of nucleotides to availability of **energy**! If limited O2 (decreased ATP), decrease activity of DHODH and get decreased synthesis of nucleotides, which is good because don't want to be making nucleotides when you don't have enough energy)

Answer 2

Multifunctional, but adds **orotate** to **PRPP** then converts that to **UMP** UMPS is also what targets and activates **5FU** (chemotherapeutic drug): give cells 5FU, and **UMPS places 5FU onto PRPP** (as if 5FU was a base like orotate) to make **active form of 5FU drug** which is like a nucleotide Note: cancer cells can be resistant to 5FU if they don't have UMPS since UMPS is needed to get active form of 5FU

Answer 3

Since cancer cells **upregulate the pyrimidine synthesis pathway** compared to normal cells (which still do have some of this pathway), 5FU acts mostly to inhibit UMP synthesis in cancer cells

Answer 4

**Carbamoyl phosphate** is the product of step 1 of pyrimidine synthesis and is necessary for urea cycle to proceed (ornithine --\> citrulline) Significance is that if **problem with urea cycle**, will get **backup of carbamoyl phosphate** in the mitochondria --\> carbamoyl phosphate will leak out into cytoplasm and cause **increase in pyrimidine biosynthesis** pathway --\> causes more uracil production, and get **uracil in blood**

Answer 5

**Inhibition** occurs by end products **UTP** and **CTP** on initial step, and **UMP** inhibits its own formation **Activation** by **ATP** and **PRPP** on first step

Answer 6

Can't make UMP because **deficiency** of **OPRT** (orotate-phosphoribosyl transferase; step 5) and/or **ODC** (orotidine monophosphate decarboxylase; step 6) (both part of **UMPS**) Causes accumulation of **orotic acid (orotate)**, which is excreted in urine, also causes anemia, retarded growth, immunodeficiency Treatment: include uridine (nucleoside) in **diet** to increase UMP levels, which allows you to make pyrimidines again AND gives negative feedback on system so don't get buildup of orotic acid Very rare, only 17 cases worldwide

Answer 7

Remember, we use **nucleosides** for **salvage** pathway of **pyrimidines**, NOT bases! If we gave uracil, we'd have to attach it to PRPP, and that's the step that is defective!

Answer 8

Pyrimidine rings fully degraded to soluble structures! (unlike purines that make uric acid)

Answer 9

**Salvage** pathway dominates over de novo synthesis for **pyrimidines** Food pyrimidines are absorbed as their nucleosides --\> pyrimidine nucleoside + **PRPP** (via pyrimidine phosphoribosyl transferase) --\> UMP?? Note: since pyrimidines are recycled at the level of the nucleotide you shouldn't have to react with PRPP...looks like Uridine has to react with PRPP (wikipedia) but Cytidine and deoxyThymidine probably don't and are just used as they are...

Answer 10

**Purines**: de novo synthesized **onto** **PRPP**; first nucleotide made is **IMP**; salvaged through **base**; degraded to **uric acid** **Pyrimidines**: de novo synthesize rings first **then add to PRPP**; first nucleotide made is **UMP**; salvaged through **nucleoside**; degraded to AA, CO2, NH3

Answer 11

Fe-containing multifunctional enzyme with redox-active thiol groups that can transfer electrons during reduction reactions RNR reduces **NDP** (ribonucleotide) **--\> dNDP** (deoxyribonucleotide) After catalyzing reaction, RNR needs to be re-oxidized by thioredoxin (note ultimate source of electrons is NADPH) Note that at this stage, have 2 phosphates attached (XX DI phosphate) Also, dATP inhibits RNR just because of feedback inhibition

Answer 12

**Phosphorylation** of dNDPs (duh...di to tri-phosphate) catalyzed by **nucleoside diphosphate kinase**

Answer 13

Thymine is a **pyrimidine** that is only in **DNA**, and is different from uracil just by 1 methyl group (thymine = 5-methyluracil)

Answer 14

**dUMP** + **THF** --\> dTMP + dihydrofolate (DHF) (via thymidylate synthase)

Answer 15

Because DNA polymerase **isn't good at distinguishing** between dUTP and dTTP but we don't want dUTP incorporated into DNA! Don't want lots of dUTP around!

Answer 16

DHF + **NADPH** + H+ --\> THF + NADP+ (via **dihydrofolate reductase**) The enzyme dihydrofolate reductase is very important because this whole cycle is required for DNA synthesis to occur!

Answer 17

**Chemotherapeutic** drugs and **antibiotics** target and inhibit dihydrofolate reductase so you can't regenerate THF from DHF This means you can't continue doing DNA synthesis (because don't have thymidine nucleotide)! These drugs can be used to kill ectopic pregnancy embryo

Answer 18

**THF** and **SAM** are carriers of methyl groups, but **serine** is the original source of the methyl group

Answer 19

5-fluorodeoxyuridylate When 5FU is given as a drug, remember UMPS adds a ribose sugar onto it to make it like a nucleotide, and FdUMP is the "nucleotide" it is converted to FdUMP is a **suicide inhibitor** of **thymidylate synthase**, so prevents thymidine synthesis and thus DNA synthesis Enzyme thymidylate synthase tries to react with FdUMP, but FdUMP has a F where UMP just has an H, and thymidylate synthase trying to react with that C-F bond inactivates the thymidylate synthase

Answer 20

Because 5FU inhibits nucleotide synthesis, specifically it **inhibits thymidine synthesis.** However, **that in turn inhibits DNA synthesis** since you need thymidine to synthesize DNA

Answer 21

DHF analogs that competitively bind to and **inhibit DHF reductase** (can't re-form THF) Ex: aminopterin, methotrexate (amethopterin)

Answer 22

Antibiotic Antifolate that binds better to **bacterial** **DHF reductase** (can't re-form THF) so is used as antibiotic

Answer 23

Folate is natural form Folic acid is synthetic form Does 1 carbon transfers Metabolic roles: synthesis of **thymidine** (using methylene-THF) and **purines** (which we didn't talk about), synthesis of methyl donor S-adenosylmethionine (**SAM**), conversion of **homocysteine to methionine** (N5-methyl-THF)

Answer 24

No, only **bacteria** can so we have to eat folate in our diet to get it

Answer 25

Since bacteria use para-amino benzoid acid (PABA) as a precursor for folic acid synthesis, we can give sulfonamide, an analog of PABA to **inhibit folic acid synthesis in bacteria** Inhibiting folic acid synthesis in bacteria eventually causes bacteria to not be able to make thymidine, so get DNA synthesis inhibition in bacteria

Answer 26

Need vitamin B12 and folic acid in the reaction where you take methyl group from THF, transfer it to **methionine** to **make SAM** and turn homocysteine --\> methionine Homocysteine --\> methionine (this rxn is very important because need SAM and THF to do make purines and dTMP (DNA synthesis!!!))

Answer 27

Caused by inadequate intake or for B12 also could be due to poor absorption due to lack of intrinsic factor Low folic acid can cause neural tube defects Low vitamin B12 can cause megaloblastic anemia, pernicious anemia, paresthesias, peripheral neuropathy, irritability, dementia, depression, psychosis

Answer 28

Inhibit nucleotide synthesis or inhibit DNA synthesis directly Used as **cancer drugs** **Antifolates** (deplete cells of thymidine), **5FU**, 6-MP, araC, 5-azacytidine, AZT, acyclovir, other pyrimidine or purine analogs Like 5FU, are activated in specific cell type (cells growing rapidly)

Answer 29

Is an antimetabolite (**purine analog**) that inhibits amido-phosphoribosyl-transferase (enzyme that catalyzes committed step in purine de novo synthesis, NOT APRT!) Converted to nucleotide analog by HGPRT in salvage pathway: 6-MP + PRPP --\> 6-MP-ribose-phosphate + PPi Used for childhood leukemia Note: relies on salvage pathway to be activated but **inhibits de novo pathway** Other derivatives exist with similar effects: Imuran (Azathioprine) used as immunosuppressant

Answer 30

Another nucleotide analog (anti-metabolite) that has same base but slightly different ribose sugar Gets **incorporated** into DNA but because of slightly different structure, **inhibits DNA chain synthesis**

Answer 31

Another anti-metabolite (similar to cytidine) thats base has nitrogen group instead of carbon When you try to make thymidine from cytidine (I thought you made T from U...) by methylating that carbon, realize it's nitrogen instead so can't methylate! Used to treat myelodysplastic syndrome (inhibits DNA synthesis) Used in basic science to decrease DNA methylation in cultured cells

Answer 32

Nucleotide **(pyrimidine) analog** that was one of the first HIV anti-retroviral drugs Instead of OH group, has N3 (azide) group Inhibits viral polymerase, **inhibits reverse-transcription**

Answer 33

Acycloguanisine (**purine analog**) is like guanine but ribose sugar ring is actually open! Must be **phosphorylated by viral thymidine kinase** to be activated (our kinases can't) Activated acyclovir is a **DNA polymerase inhibitor**

Answer 34

Hypoxanthine is more soluble, so is just excreted!