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M1 Biochemistry > Exam 2- Nucleotide Metabolism > Flashcards

Exam 2- Nucleotide Metabolism Flashcards

1
Q

For de novo purine synthesis, named the 3 amino acids that are required

A

Glutamine, Aspartate, and Glycine

  • donate nitrogen groups
  • Glycine both nitrogen and carbon
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2
Q

Name the cofactor (vitamin derivative) that serves as a carbon donor in purine synthesis.

A

Tetrahydrofolate (N10-formal-H4-folate)

- donates formyl group

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3
Q

Name the ribose compound on which purines are constructed.

A

Phosphoribosylamine

- derivate of PRPP (5-phosphoribosyl 1-pyrophosphate)

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4
Q

Name the rate limiting enzyme in purine synthesis, and its positive and negative allosteric effectors.

A

PRAT (phosphoribosylamine transferase)
- Converts PRPP into phosphoribosylamine using an amino group donated by glutamine

Allosteric activator: PRPP (substrate)
Allosteric inhibitors: IMP, GMP, and AMP (end pathway products)

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5
Q

Name a compound other than ATP that is used in the biosynthesis, which is common for fatty aid synthesis, urea cycle, purine and pyrimidine synthesis.

A

NAD+

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6
Q

What is the common branch point precursor fro AMP and GMP synthesis?

A

IMP (inosine monophosphate)

- formed after 9 sequential enzymatic steps from phosphoribosylamine

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7
Q

Name two rate limiting steps and their allosteric effectors in purine synthesis (after IMP production)?

A

Adenylosuccinate synthase

  • forms adenylosuccinate from IMP using GTP and aspartate
    • effector: GTP
  • pathway forms AMP

IMP dehydrogenase

  • forms Xanthine monophosphate (XMP) using NAD+
    • Effector: ATP
  • pathway forms GMP
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8
Q

Name two salvage pathway enzymes for Purines

A

1) Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)
- Forms IMP from Hypoxanthine and PRPP
- Forms GMP from Guanine and PRPP

2) Adenine phosphoribosyltransferase (APRT)
- Forms AMP from Adenine and PRPP

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9
Q

Name a cell type that is solely dependent upon salvage pathway enzymes for nucleotide synthesis

A

Erythrocytes- cannot perform de novo synthesis

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10
Q

Name the nucleotide from which tetrahydrobiopterine is synthesized.

A

GTP

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11
Q

Name the immediate precursor of Uric acid

A

Xanthine

- formed form Hypoxanthine or Guanine (nitrogenous bases)

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12
Q

Name the enzyme that convert is Xanthine into uric acid

A

Xanthine oxidase

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13
Q

Explain the reason for excess uric acid production in gout patient.

A

Defects in regulation of purine pathway can lead to overproduction of purine nucleotides through de novo pathway due to increased PRPP availability- increased PRPP synthetase activity or decreased HGPRT activity

  • Consequence is overproduction of uric acid (leads to urate crystals in joints of extremities —> arthritis)
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14
Q

Name drugs that are used for treatment of gout.

A

Allopurinol
Alloxanthine
Cholchicine

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15
Q

Explain the reason for gout in Von Gierke’s patients.

A

Glucose-6-phosphatase activity is lacking leading to excess glucose-6-phosphate that enters the HMP shunt producing excess Ribose-5-phosphate —> results in excess de novo purine synthesis.
- Consequence is overproduction of uric acid

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16
Q

Name two enzymes that are deficient in combined immunodeficiency.

A 
Adenosine deaminase (ADA) deficiency 
- causes defective T cell and B cell immunity

Purine nucleoside phosphorylase (PNP) deficiency
- causes T-cell dysfunction

Both cause dATP to accumulate and inhibits ribonucleotide reductase activity —> DNA synthesis inhibited in T-cells and B-cells

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17
Q

What causes Lesch-Nyhan syndrome?

A

X-linked Hyperuricemia

Due to loss of HGPRT enzyme —> Hypoxanthine and guanine not salvaged, PRPP levels high, AMP and GMP levels low —> increased de novo synthesis —> increased uric acid synthesis

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18
Q

Carbamoyl phosphate synthase II, which is involved in pyrimidine synthesis, is located in ___________ of the cell.

A

Cytosol

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19
Q

Name two amino acids that are required for pyrimidine synthesis.

A

Glutamine
- Donates an amino group

Aspartate
- Donates carbon as well as a nitrogen group

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20
Q

True or False. Pyrimidine synthesis involves cytosolic as well as mitochondrial compartments

A

True.

All enzymes including CAD and UMP synthase are cytosolic enzymes except Dihydroorotase dehydrogenase is a mitochondrial enzyme

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21
Q

In pyrimidine synthesis the ring structure is first formed and then the nitrogenous base is transferred to PRPP

A

True

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22
Q

In purine synthesis, it is the PRPP molecule on which the purine ring is constructed.

A

False

The purine ring (Hypoxanthine = nitrogenous base w/o ribose unit) is built upon Phosphoribosylamine

23
Q

OMP is converted to UMP by the enzyme

A

UMP synthase

- byproduct CO2

24
Q

CTP is directly synthesized from the nucleotide ________.

A

UTP

- catalyzed by the enzyme CTP synthase using an amino group from glutamine and ATP

25
Q

Name the enzyme that is deficient in hereditary orotic aciduria.

A

UMP synthase deficiency

- Tx: uridine

26
Q

Name an amino acid that serves as a carbon source for pyrimidine synthesis.

A

Aspartate

- Bicarbonate is also a carbon donor

27
Q

Ribonucleotide reductase converts NDP into dNDP, and uses the coenzyme ____________.

A

Thioredoxin

  • is a cysteine containing thiol (-SH) protein
  • engages in reduction/oxidation coupled reactions
  • during reaction it goes from its reduced form (-SH) to its oxidized form (-S-S)
  • Ribonucleotide reductase is activated by ATP and inhibited by dATP
28
Q

For thioredoxin reductase activity, the reducing compound that is required is _________.

A

NADPH

  • catalyze by Thioredoxin reductase
  • Thioredoxin is regenerated to its reduced form (-SH)
29
Q

Name two enzymes that are required for deoxynucleotide synthesis.

A

Ribonucleotide reductase
- Converts NDP into dNDP using coenzyme thioredoxin

Nucleotide disphosphate kinase
- Uses ATP to catalyze dNDP —> dNTP

30
Q

dCMP is converted into dUMP by the _____________ enzyme.

A 
dCMP deaminase (rate limiting enzyme)
- After dCDP is converted into dCMP by phosphatase
31
Q

dTMP is converted into dTTP by the enzyme _________.

A 
Thymidylate kinase (TK)
dTTP in excess inhibits rate limiting enzyme dCMP delaminates
32
Q

The conversion of dihydrofolate to tetrahydrofolate during dTMP synthesis is catalyze by the enzyme _____________.

A

DiHydroFolate Reducatase (DHFR) using NADPH

33
Q

Name the four nucleotide triphosphates (dNTP) that are required for DNA synthesis.

A

dATP, dGTP, dCTP, and dTTP

34
Q

During S phase of cell proliferation, name the enzymes that are up regulated in

  • Purine metabolism
  • Pyrimidine metabolism
  • enzymes unique to DNA metabolism
A

Purine metabolism:
- PRPP amidotransferase and IMP dehydrogenase enzymes are upregulated

Pyrimidine metabolism:
- CAD, UMP synthase, and CTP synthase

DNA:
- ribonucleotide reductase, dCMP deaminase, thymidylate synthase, and thymidylate kinase enzymes

35
Q

Name the respective vitamins that are required for the synthesis of NAD+, FAD+, and CoA

A
  • note these 3 are cofactors derived from nucleic acids:

NAD+:
Vitamin B3 niacin (serves as the source of nicotinamide)

FAD:
Riboflavin (vitamin B2)

CoA:
Pantothenate (one of vitamin B complex)

36
Q

Name the Amino Acid that is required for CoA synthesis.

A

Cysteine

37
Q

In CoA, the “business end” of the molecule has the following group ________

A

Thiol group

- provided by the Cysteine

38
Q

Name 3 anti leukemic drugs

A

6-mercaptopurine (6MP, an analog of purine)

  • Transferred to PRPP to produce 6MP-ribonucleotide monophosphate, which is a negative effector of PRPP amidotransferase and adenylosuccinate synthase
  • Results in arrest of de novo DNA synthesis

5-Fluoruouracil (FU an analog of Pyrimidine)

  • Converted into FdUMP (analog of dUMP), which is an inhibitor of thymidylate synthase
  • results in arrest of dTMP synthesis

Cytosine arabinoside (Ara C)

  • Has an arabinose carbohydrate moiety instead of usual 2’-deoxy ribose of dNTP
  • DNA polymerization terminates prematurely
39
Q

6-mercaptopurine

A

6-mercaptopurine (6MP, an analog of purine)

  • Transferred to PRPP to produce 6MP-ribonucleotide monophosphate, which is a negative effector of PRPP amidotransferase and adenylosuccinate synthase
  • Results in arrest of de novo DNA synthesis
40
Q

5-Fluorouracil

A

5-Fluoruouracil (FU an analog of Pyrimidine)

  • Converted into FdUMP (analog of dUMP), which is an inhibitor of thymidylate synthase
  • results in arrest of dTMP synthesis
41
Q

Cytosine arabinoside (Ara C)

A

Cytosine arabinoside (Ara C)

  • Has an arabinose carbohydrate moiety instead of usual 2’-deoxy ribose of dNTP
  • DNA polymerization terminates prematurely
42
Q

Compounds that inhibit tumor cell growth

A

Hydroxyurea- Inhibits DNA synthesis by inhibiting ribonucleotide reductase and conversion of NDP to dNDP

Tiazofurin- NAD+ analog.

43
Q

Describe the mechanism of actin of TAD

A

Tiazofurin adenine dinucleotide inhibits IMP dehydrogenase (rate limiting step in GTP synthesis)
Results in reduction of cellular GTP synthesis

44
Q

Describe the mode of action of AZT

A

Azido-3’-deoxythymidine (AZT)-

Antiviral
A Pyrimidine Analog which is converted into AZT-triphosphate and incorporated into DNA
Cannot polymerize further since 3’-azido group cannot engage in phosphodiester formation
Viral RNA dependent DNA polymerase (reverse transcriptase) is more sensitive than host cell DNA dependent DNA polymerase

45
Q

Describe the mode of action of Acyclovir.

A

Acycloguanosine (Acyclovir)

Antiviral
Is converted into an active form by viral thymidine kinase and eventually converted into acycloguanosine triphosphate and incorporated into viral DNA
Results in termination of polymerization, since it does not have the 3’-hydroxyl-group available for polymerization

46
Q

Name two glutamine antagonists

A

Azaserine and DON

  • inhibit many enzymes that utilize glutamine as the amino group donor:
    1. Purine de novo synthesis
    2. Synthesis of IMP from GMP
    3. Cytotoxic carbamoyl phosphate synthesis
    4. Synthesis of CTP from UTP
    5. Synthesis of NAD+
  • Extremely cytotoxic and not very clinically useful
47
Q

Describe the mode of action of methotrexate (MTX).

A

An analog of folate
Inhibits dihydrofolate reductase activity, preventing the conversion of dihydrofolate into tetrahydrofolate
Reduces dTMP production from dUMP

48
Q

Name the two types of drug resistance and the mechanism of resistance.

A
  1. Specific drug resistance
    a) MTX resistance can arise form alterations in folate transporters
    b) marked amplification of dihydrofolate reductase gene
    c) Generation of mutant dihydrofolate reductase gene
  2. Multidrug-resistance
    a) Synthesis of proteins MDR1 (glycoproteins) or MRP (multidrug resistance protein)
    b) These membrane bound proteins are pumps that remove drugs from cells
49
Q

The nucleotide analog that is used in DNA sequencing is ______________.

A

ddNTP

  • dideoxy analog that stops the polymerization reaction randomly
  • generates various lengths of DNA segments
50
Q

The DNA polymerase used in the PCR reaction is obtained from the organism _____________.

A

Thermus aquaticus

- Thermophilic bacterium that thrives at extremely high temperatures (180 degrees C)

51
Q

Name the components that are required for gene cloning.

A
  • Restriction enzymes- Digest, cleave the DNA of interest
  • DNA Ligase - inserts the DNA of interest into the cloning vector (plasmid)
  • Cloning vector can then be transformed into bacteria (by CaCl2 or heat shock method)
  • Recombinant bacteria are then selected by antibiotic sensitivity towards ampicillin or kanamycin
52
Q

What is the role of restriction enzymes in molecular cloning?

A

To cleave out the area of interest from DNA

53
Q

Name few recombinant screening techniques.

A
  1. Blue/white color method (beta galactosidase assay used) white- recombinant bacteria, blue- nonrecombinant
  2. Restriction enzyme fragment length polymorphism (RFLP)

M1 Biochemistry (17 decks)

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