Nucleotide Metabolism Lecture 1-4 - Dr. Venk

Question 1

For de novo synthesis of purine, name three amino acids that are required

Answer 1

glutamine (nitrogen)
aspratate (nitrogen)
glycine (carbon and nitrogen)

Question 2

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

Answer 2

THF

Question 3

Name the ribose compound on which purines are constructed

Answer 3

Ribose-5-phosphate –> 5-phosphoribosyl 1-pyrophosphate (PRPP)

Question 4

Name the rate limiting enzyme in purine synthesis and it’s positive and negative allosteric effectors

Answer 4

Enzyme: phosphoribosylamine Transferase (PRAT)

(+): PRPP
(-): AMP, IMP, GMP

Question 5

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

Answer 5

Bicarbonate!

Question 6

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

Answer 6

IMP

Question 7

Name two rate limiting enzymes and their allosteric effectors in purine synthesis (After IMP production)

Answer 7

Adenylosuccinate synthase: (+) GTP

IMP dehydrogenase: (+) ATP

Question 8

Name two salvage pathway enzymes

Answer 8

1) hypoxanthine-guanine phosphoribosyl transferase (HGPRT)

2) APRT

Question 9

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

Answer 9

erythrocytes

Question 10

Name the immediate precursor of uric acid

Answer 10

Xanthine

Question 11

Name the enzyme that converts xanthine into uric acid

Answer 11

Xanthine Oxidase

Question 12

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

Answer 12

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.

Question 13

Name the nucleotide from which tetrahydrobiopterin is synthesized

Answer 13

GTP via cyclohydrolase

Question 14

Name two drugs that are used for treatment of gout

Answer 14

Allopurinol and Alloxanthine (also cholchine)

Question 15

Explain the reason for gout in Von Gierke’s patients

Answer 15

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.

Question 16

Name two enzymes that are deficient in combined immunodeficiency

Answer 16

Adenosine deaminase (ADA) deficiency leads to defective T-cell and B-cell immunity

Purine nucleoside phosphorylase (PNP) deficiency leads to T-cell dysfunctions.

Question 17

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

Answer 17

cytosol

Question 18

Name two amino acids that are required for pyrimidine synthesis

Answer 18

Glutamine and aspartate

Question 19

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

Answer 19

True

Question 20

True or False: In pyrimidine synthesis, the ring structure is first formed and then the nitrogenous base is transferred to PRPP

Answer 20

True. Ring first then add PRPP

Question 21

True or False: In purine synthesis, it is the PRPP molecule on which the purine ring is constructed

Answer 21

TRUE. PRPP starts first

Question 22

OMP is converted to UMP by the enzyme _______.

Answer 22

UMP synthase

Question 23

CTP is directly synthesized from the nucleotide ________.

Answer 23

UTP

Question 24

Name the enzyme that is deficient in hereditary orotic aciduria

Answer 24

UMP synthase

Question 25

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

Answer 25

Aspartate

Question 26

Ribonucleotide reductase converts NDP into dNDP and uses the coenzyme _________.

Answer 26

Thioredoxin

Question 27

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

Answer 27

NADPH

Question 28

Name two enzymes that are required for deoxynucleotide synthesis

Answer 28

ribonucleotide reductase and thioredoxin reductase

Question 29

dCMP is converted into dUMP by the ________ enzyme

Answer 29

CMP deaminase

Question 30

dTMP is formed from dUMP by _________ which uses the cofactor _____.

Answer 30

thymidylate synthase ; N5-N10-Metylene THF

Question 31

The conversion of dihydrofolate to tetrahydrofolate during dTMP synthesis is catalyzed by the enzyme _____.

Answer 31

dihydrofolate reductase

Question 32

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

Answer 32

dATP, dGTP, dCTP, and dTTP.

Question 33

During the S phase of cell proliferation, name the enzymes that are upregulated in purine metabolism

Answer 33

PRPP amindotransferase | IMP dehydrogenase

Question 34

During the S phase of cell proliferation, name the enzymes that are upregulated in pyrimidine metabolism.

Answer 34

UMP synthase, CTP synthase, and CAD

Question 35

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

Answer 35

FAD+ : Vitamin B2 (riboflavin) NAD+ : Vitamin B3 (niacin/nicatinamide) CoA : Vitamin B5 (pantothenate)

Question 36

Name the amino acid that is required for CoA synthesis

Answer 36

Cysteine

Question 37

In CoA, the "Business end" of the molecule has the following group: __________

Answer 37

thiol

Question 38

Name two anti leukemic drugs.

Answer 38

6MP, FU, and AraC

Question 39

Describe the mechanism of action of 6-MP.

Answer 39

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.

Question 40

Describe the mechanism of action of 5-FU

Answer 40

Converted to FdUMP, an analog of dUMP. It is an inhibitor of thymidylate synthase, stopping dTMP synthesis.

Question 41

Describe the mechanism of action of AraC

Answer 41

With an arabinose carbohydrate moiety instead of the 2'-deoxy ribose of dNTP, DNA polymerizaiton terminates prematurely.

Question 42

Name a compound that inhibits tumor cell growth.

Answer 42

Hydroxyurea

Question 43

Describe the mechanism of action of TAD

Answer 43

inhibits IMP dehydrogenase b/c it decreases GTP availability

Question 44

Describe the mode of action of AZT.

Answer 44

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.

Question 45

Describe the mode of action of Acyclovir.

Answer 45

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.

Question 46

Name two glutamine antagonists

Answer 46

DON and Azaserine

Question 47

Describe the mode of action of MTX

Answer 47

MTX inhibits dihydrofolate reductase activity, preventing the conversion of dihydrofolate into tetrahydrofolatte. This reduces TMP production.

Question 48

Name the two types of drug resistance and the mechanisms of resistance

Answer 48

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

Question 49

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

Answer 49

didoxy analogs (ddNTP)

Question 50

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

Answer 50

Thermus aquaticus

Question 51

Name the components that are required for gene cloning

Answer 51

1) DNA template 2) Primers 3) normal dNTPS 4) DNA polymerase @ 37 degrees

Question 52

Name the steps of gene cloning.

Answer 52

1) Denaturation (melting) @ 95 degrees C 2) Annealing (Cooling) @ 44-55 degrees C 3) Synthesis @ 75 degrees C

Question 53

What is the role of restriction enzymes in molecular cloning?

Answer 53

To cut the DNA into vectors with similar endings so that they may be annealed back in any order/fashion

Question 54

Describe the process involved in molecular cloning.

Answer 54

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

Question 55

Name a few restriction enzymes.

Answer 55

EcoR1, Bgal1, HACIII

Question 56

Name a few recombinant screening techniques

Answer 56

- antibiotic sensitivity toward ampicillin or kanamycin | - marker genes like beta-lactamase or beta-galactosidase (blue/white color selection method)

Question 57

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.

Answer 57

c) adenosine triphosphate (ATP) and flavin mononucleotide (FMN). (riboflavin)

Question 58

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.

Answer 58

a) nicotinamide adenine dinucleotide (niacin) and adenosine triphosphate (ATP)

Question 59

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)

Answer 59

b) ATP and cysteine pantothenate, 4 ATP, and Cysteine]

Question 60

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

Answer 60

e) sequencing