Nucleotide Metabolism Lecture 1-4 - Dr. Venk Flashcards
For de novo synthesis of purine, name three amino acids that are required
glutamine (nitrogen)
aspratate (nitrogen)
glycine (carbon and nitrogen)
Name the cofactor (vitamin derivative) that serves as a carbon donor in purine syntehesis
THF
Name the ribose compound on which purines are constructed
Ribose-5-phosphate –> 5-phosphoribosyl 1-pyrophosphate (PRPP)
Name the rate limiting enzyme in purine synthesis and it’s positive and negative allosteric effectors
Enzyme: phosphoribosylamine Transferase (PRAT)
(+): PRPP
(-): AMP, IMP, GMP
Name a compound other than ATP that is used in the biosynthesis, which is common for fatty acid synthesis, urea cycle, purine and pyrimidine synthesis
Bicarbonate!
What is the common branch point precursor for AMP and GMP synthesis?
IMP
Name two rate limiting enzymes and their allosteric effectors in purine synthesis (After IMP production)
Adenylosuccinate synthase: (+) GTP
IMP dehydrogenase: (+) ATP
Name two salvage pathway enzymes
1) hypoxanthine-guanine phosphoribosyl transferase (HGPRT)
2) APRT
Name a cell type that is solely dependent upon salvage pathway enzymes for nucleotide synthesis
erythrocytes
Name the immediate precursor of uric acid
Xanthine
Name the enzyme that converts xanthine into uric acid
Xanthine Oxidase
Explain the reason for excess uric acid production in gout patients.
Increased PRPP synthetase activity or decreased HGPRT activity leads to overproduction of purine nucleotides due to the increased availability of PRPP. This leads to an overproduction of uric acid.
Name the nucleotide from which tetrahydrobiopterin is synthesized
GTP via cyclohydrolase
Name two drugs that are used for treatment of gout
Allopurinol and Alloxanthine (also cholchine)
Explain the reason for gout in Von Gierke’s patients
Decreased glucose 6-phosphatase activity leads to excess glusose 6-phosphate, which enters the HMP shunt and creates excess ribose-5-Phosphate. This leads to excess uric acid production.
Name two enzymes that are deficient in combined immunodeficiency
Adenosine deaminase (ADA) deficiency leads to defective T-cell and B-cell immunity
Purine nucleoside phosphorylase (PNP) deficiency leads to T-cell dysfunctions.
Carbamoyl phosphate synthase II, which is involved in pyrimidine synthesis is located in the _____ part of the cell.
cytosol
Name two amino acids that are required for pyrimidine synthesis
Glutamine and aspartate
True or False: Pyrimidine synthesis involves cytosolic as well as mitochondrial compartments
True
True or False: In pyrimidine synthesis, the ring structure is first formed and then the nitrogenous base is transferred to PRPP
True. Ring first then add PRPP
True or False: In purine synthesis, it is the PRPP molecule on which the purine ring is constructed
TRUE. PRPP starts first
OMP is converted to UMP by the enzyme _______.
UMP synthase
CTP is directly synthesized from the nucleotide ________.
UTP
Name the enzyme that is deficient in hereditary orotic aciduria
UMP synthase
Name an amino acid that serves as a carbon source for pyrimidine synthesis
Aspartate
Ribonucleotide reductase converts NDP into dNDP and uses the coenzyme _________.
Thioredoxin
For thioredoxin reductase activity, the reducing compound that is required is ________.
NADPH
Name two enzymes that are required for deoxynucleotide synthesis
ribonucleotide reductase and thioredoxin reductase
dCMP is converted into dUMP by the ________ enzyme
CMP deaminase
dTMP is formed from dUMP by _________ which uses the cofactor _____.
thymidylate synthase ; N5-N10-Metylene THF
The conversion of dihydrofolate to tetrahydrofolate during dTMP synthesis is catalyzed by the enzyme _____.
dihydrofolate reductase
Name the four nucleotide triphosphates (dNTP) that are required for DNA synthesis
dATP, dGTP, dCTP, and dTTP.
During the S phase of cell proliferation, name the enzymes that are upregulated in purine metabolism
PRPP amindotransferase
IMP dehydrogenase
During the S phase of cell proliferation, name the enzymes that are upregulated in pyrimidine metabolism.
UMP synthase, CTP synthase, and CAD
Name the respective vitamins that are required for the synthesis of FAD+, NAD+, and CoA
FAD+ : Vitamin B2 (riboflavin)
NAD+ : Vitamin B3 (niacin/nicatinamide)
CoA : Vitamin B5 (pantothenate)
Name the amino acid that is required for CoA synthesis
Cysteine
In CoA, the “Business end” of the molecule has the following group: __________
thiol
Name two anti leukemic drugs.
6MP, FU, and AraC
Describe the mechanism of action of 6-MP.
6-MP is transferred to PRPP to produce 6MP-ribonucleotide monophosphate, which is a negative effector of PRPP amidotransferase and adenylatosuccinate synthase. This results in the arrest of the de novo synthesis of DNA.
Can be prolonged by coadministering allopurinol.
Describe the mechanism of action of 5-FU
Converted to FdUMP, an analog of dUMP. It is an inhibitor of thymidylate synthase, stopping dTMP synthesis.
Describe the mechanism of action of AraC
With an arabinose carbohydrate moiety instead of the 2’-deoxy ribose of dNTP, DNA polymerizaiton terminates prematurely.
Name a compound that inhibits tumor cell growth.
Hydroxyurea
Describe the mechanism of action of TAD
inhibits IMP dehydrogenase b/c it decreases GTP availability
Describe the mode of action of AZT.
A pyrimidine analog that is converted into AZT-triphosphate and gets incorporated into DNA, but cannot polymerize further since no phosphodiester bonds can form.
Affects viral RNA dependent DNA polymerase b/c they are 100x more sensitive to AZT.
Describe the mode of action of Acyclovir.
Acyclovir is converted into its active form by viral thymidine kinase, which is converted into acycloguanosine triphosphate and incorporated into viral DNA. This results in the termination of polymerization b/c there is no deoxyribose available for polymerization.
Name two glutamine antagonists
DON and Azaserine
Describe the mode of action of MTX
MTX inhibits dihydrofolate reductase activity, preventing the conversion of dihydrofolate into tetrahydrofolatte. This reduces TMP production.
Name the two types of drug resistance and the mechanisms of resistance
Specific Drug Resistance and Multi drug resistance
Specific Drug Resistance:
a) MTX resistance
b) Amplification of DHFR (dihydrofolate reductase)
c) DHFR isoform resistance to MTX
Multi-drug resistance
a) synthesis of MDR1 (glycoprotein) or MRP
The nucleotide analog that is used in DNA sequencing is _________.
didoxy analogs (ddNTP)
The DNA polymerase used in PCR reaction is obtained from the organism
Thermus aquaticus
Name the components that are required for gene cloning
1) DNA template
2) Primers
3) normal dNTPS
4) DNA polymerase @ 37 degrees
Name the steps of gene cloning.
1) Denaturation (melting) @ 95 degrees C
2) Annealing (Cooling) @ 44-55 degrees C
3) Synthesis @ 75 degrees C
What is the role of restriction enzymes in molecular cloning?
To cut the DNA into vectors with similar endings so that they may be annealed back in any order/fashion
Describe the process involved in molecular cloning.
1) Use restriction enzymes to create vectors of interest and cut open the cloning vector
2) Ligate the DNA of interest into plasmid
3) Transform bacteria by CaCl2 or heat shock method
Name a few restriction enzymes.
EcoR1, Bgal1, HACIII
Name a few recombinant screening techniques
- antibiotic sensitivity toward ampicillin or kanamycin
- marker genes like beta-lactamase or beta-galactosidase (blue/white color selection method)
FAD+ is formed from…
a) nicotinamide adenine dinucleotide and adenosine triphosphate (ATP)
b) beta alanine and beta-aminoisobutyrate
c) adenosine triphosphate (ATP) and flavin mononucleotide (FMN).
d) fiboflavin and deoxyguanosine triphosphate (dGTP)
e) riboflavin and ADP-ribose.
c) adenosine triphosphate (ATP) and flavin mononucleotide (FMN). (riboflavin)
NAD+ is formed from…
a) nicotinamide adenine dinucleotide and adenosine triphosphate (ATP)
b) beta alanine and beta-aminoisobutyrate
c) adenosine triphosphate (ATP) and flavin mononucleotide (FMN).
d) fiboflavin and deoxyguanosine triphosphate (dGTP)
e) riboflavin and ADP-ribose.
a) nicotinamide adenine dinucleotide (niacin) and adenosine triphosphate (ATP)
Coenzyme A is synthesized in part from
a) ATP and niacin
b) ATP and cysteine
c) CTP (cytidine triphosphate)
d) ATP and aspartate
e) GTP (guanosine triphosphate)
b) ATP and cysteine
pantothenate, 4 ATP, and Cysteine]
PCR (polymerase chain reaction) might be used to amplify DNA for direct use in
a) a yeast expression system
b) a eukaryotic expression system
c) a bacterial expression system
d) a microarray experiment
e) sequencing
e) sequencing
