Week 4: Nucleotide Metabolism II Flashcards

1
Q

What does ribonucleotide reductase do?

A

Converts NDP (nucleotide diphosphate) to dNDP (deoxynucleotide diphosphate)

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2
Q

What are the kinetic requirements for nucleotides to enter the synthesis pathway?

A

Low Km, high Vmax

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3
Q

Why is it critical to keep nucleotide levels (C, G, A and T) at low and relatively equal levels?

A

To ensure accuracy in nucleotide selection for metabolism

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4
Q

How can “the wrong” nucleotide be synthesized?

A

Though the Km is very high and Vmax is very low for these situations, if there is an imbalanced amount of one nucleotide, it can oversaturate and begin to enter the synthesis pathway

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5
Q

What is the overall function of the conversion of NDP to dNDP?

A

To create dNDPs for DNA synthesis

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6
Q

Which NDP to dNDP conversion is unusual, and how does it occur?

A

dTDP is unusual because you start with dUDP, and have to add an H group to it to convert it to dTDP (requires the removal of -OH group, and then the reduction by hydrogen to make it a 2’ deoxynucleotide)

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7
Q

Where do the electrons come from to deoxygenate and reduce an unusual dNDP?

A

The electron pair comes from reduced cysteine

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8
Q

Where do the hydrogens used to reduce ribonuclease reductase derive from?

A

NADH or NADPH

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9
Q

How many sites does ribonucleotide reductase have, and what are their functions?

A

There are two main regulatory sites–a secondary site for determining substrate specificity, and a primary site to turn the enzyme on/off–and an active site

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10
Q

What substrates bind to the on/off regulatory site of ribonucleotide reductase and how do they function?

A

ATP turns on the enzyme (positive feedback when there is not enough dATP) and dATP turns off the enzyme when enough has been made

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11
Q

What is the function of dUTPase in the process of forming dTMP?

A

It is a “sanitizing” enzyme that removes a pyrophosphate (beta and gamma phosphates), converting dUTP to dUMP

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12
Q

What other dNTP can be converted to dTMP and how does that occur?

A

dCTP can be deaminated to dUTP by deaminase, which can then be dephosphorylated by dUTPase, forming dUMP

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13
Q

What kind of group is added to dUMP to convert it to dTMP? What enzyme catalyzes it?

A

A methyl group at the 5C position on the 6-membered ring above the ribose molecule. Thymidylate synthase catalyzes it.

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14
Q

How is 7,8-Dihydrofolate converted back to N5N10-Methylene-tetrahydrofolate?

A

Reduction by NADPH/H+ (by dihydrofolate reductase, involved in chemotherapy) and the addition of a methyl group by the demethylation of serine to glycine

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15
Q

Generally speaking, how is AMP converted to uric acid, and why is this done?

A

AMP is converted to hypoxanthine (removal of Pi, NH3, and ribose), then xanthine (oxidation), then uric acid (another oxidation) for disposal. This is done to ensure proper balance of purines in the body, as AMP acts as an indicator of overall [NTP]

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16
Q

What are the two ribonucleotides that are usually made in excess?

A

GMP and AMP

17
Q

What is one of the ways excess uric acid causes problems in the body (normal excretion is 0.6g/24h)?

A

Because uric acid in the form of sodium urate is relatively insoluble, excess can build up in the body and joints and form crystals. Lymphocytes then phagocytose those crystals, and the inflammatory response can damage collagen in joints. Gout is caused by poor uric acid excretion or oversynthesis of purines de novo

18
Q

How does the salvaging reaction work? Why is it almost irreversible?

A

5-Phosphoribosyl-1-pyrophosphate (PRPP) is conjugated to adenine via nucleophilic attack. The two pyrophosphate moieties are then cleaved, making the reaction nearly irreversible

19
Q

Explain the guanine salvaging reaction

A

Guanine and hypoxanthine are conjugated to PRPP, forming IMP, GMP, and cleaving the pyrophosphate group to form PPi

20
Q

What is the primary deficiency involved in SCID? What occurs in these patients?

A

Adenosine deaminase (ADA), which converts adenosine to inosine. B and T lymphocytes do not develop properly in ADA patients. T lymphocyte toxicity (immune cells cannot mount a response) also results from 100-fold elevated levels of dATP

21
Q

What is the result of the dysfunction of ADA?

A

The dysfunction of Adenosine DeAminase means 2’-deoxyadenosine cannot be shunted through the disposal pathway (uric acid), and instead is converted to dATP via the deoxynucleoside kinase enzyme. ADA deficiency causes SCID, improper B- and T-lymphocyte development in ADA-deficient patients.

22
Q

Downregulation of what enzyme prevents proliferation of lymphocytes and a mounting of the immune response, as it relates to ribonucleotide/dNTP catalysis?

A

Downregulation of Adenosine DeAminase (ADA) 100-fold increase of dATP shuts down the activity of ribonucleotide reductase

23
Q

What is the pathology of Lesch-Nyhan syndrome?

A

HGPRTase deficiency (hypoxanthine-guanine phosphoribosyltransferase, which forms IMP/GMP from the conjugation of hypoxanthine/guanine and PRPP)

24
Q

What are the symptoms of Lesch-Nyhan syndrome? Why does it arise?

A

Spasticity, mental retardation, highly aggressive and self-destructive behavior. Characterized by high levels of uric acid, and is found almost exclusively in male children.

25
Q

What is the mechanism by which uric acid is increased in pts with Lesch-Nyhan?

A

PRPP and hypoxanthine are produced in excess as a result of deficient HGPRTase (usually mediates the Hypoxanthine-Guanine salvage pathway). Excess hypoxanthine moves through the catabolic degradation pathway to form uric acid instead of being salvaged.

26
Q

What are the two diseases characterized by excess uric acid? What are symptoms (and treatments, if possible) for each?

A

Gout (joint inflammation) and Lesch-Nyhan syndrome (spasticity, mental retardation, highly aggressive and self-destructive behavior). Gout can be treated with xanthine oxidase, which allows hypoxanthine and xanthine to be excreted, or with allopurinol, which acts as a competitive inhibitor of xanthine oxidase, blocking the catabolism of hypoxanthine.

27
Q

How do dNTPs relate to treatments for cancer?

A

Since most cells are in their resting state, prevention of dNTP formation can help prevent cancer cells from proliferating

28
Q

What are the classic chemotherapeutic agents that target nucleotide biosynthetic pathways?

A

Glutamine analogs (azaserine and acivicin) that block biosynthesis. However, glutamine is a central player in nitrogen metabolism. Hydroxyurea is also used because it inhibits ribonucleotide reductase

29
Q

What are the thymidylate synthase inhibitors, and what is their simple mode of inhibition?

A

Fluorouracil, an analog of thymine, inhibits thymidylate synthase directly. Methotrexate and trimethoprim are folate analogs that inhibit indirectly.

30
Q

How does fluorouracil inhibit dUMP conversion to dTMP?

A

It binds competitively at the active site of the conversion enzyme, and prevents the required hydride shift to push the molecule off by replacing the hydrogen on dUMP with a fluorine (hydrogen from enzyme cannot conjugate to FdUMP and is prevented from acting on any dUMP molecules)

31
Q

What is the stepwise synthesis of deoxyribonucleotides by ribonucleotide reductase, starting with the least common products?

A

(1) ADP binds the enzyme, signaling low [dNTP]
(2) CDP and UDP enter the enzyme and begin forming dCTP and dUDP, as the pyrimidines are least readily available
(3) dUDP is converted to dTTP, which takes the place of ADP, activating GDP binding
(4) GDP is converted to dGTP
(5) dGTP activates the conversion of ADP to dATP last, as ATP is usually the most highly available

32
Q

The enzyme (1)__________ is critical in purine recycling, converting (2)_________ and (3)___________ to (4)______ & _________ using (5)__________ as a fuel resource. It is irreversible because (6)…

A

(1) Hypoxanthine-guanine phosphoriboxyltransferase (HGPRT)
(2) hypoxanthine
(3) guanine
(4) IMP and GMP
(5) PRPP
(6) the pyrophosphate produced by the reaction is cleaved almost immediately

33
Q

Why does SCID occur from a biochemical perspective?

A

ADA deficiency prevents adenosine from entering the breakdown pathway (leading to uric acid), and causes buildup of dATP. This turns off the ribonucleotide reductase enzyme in lymphocytes specifically, preventing them from properly synthesizing B and T cells/