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Chemistry of Nucleotides

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1- Which statement best describes Xanthine ?

a) It is a direct precursor of Guanine

b) It covalently binds to Allopurinol

c) It is oxidized to form Uric acid

d) It is oxidized to form Hypoxanthine

e) It is a substrate rather than a product of the enzyme Xanthine oxidase

2- Methylated heterocyclic bases of plants include all except-

a) Caffeine

b) Theophylline

c) Theobromine

d) Thymine

e) Dimethyl Xanthine

3- Which out of the following is not an anticancer drug ?

a) 5-fluorouracil

b) 5-iodouracil,

c) 3-deoxyuridine,

d) 6-thioguanine

e) Pseudouridine

4- Feedback inhibition of pyrimidine nucleotide synthesis can occur by which of the following ?

a) Increased activity of Carbamoyl phosphate synthetase

b) Increased activity of Aspartate transcarbamoylase

c) CTP allosteric effects

d) UMP competitive inhibition

e) TTP allosteric effects

5- Which base derivative can serve as a precursor for the synthesis of two of the other pyrimidine base derivatives ?

a) Cytidine triphosphate

b) Uridine mono phosphate

c) Adenosine mono phosphate

d) deoxy thymidine mono phosphate

e) deoxy Adenosine mono phosphate

6- Purine nucleotide biosynthesis can be inhibited by which of the following ?

a) Guanosine triphosphate

b) Uridine mono phosphate

c) Adenosine mono phosphate

d)Adenosine tri phosphate

e) Inosine diphosphate

7- Which of the following contributes nitrogen atoms to both purine and pyrimidine rings ?

a) Aspartate

b) Carbamoyl phosphate

c) Carbon dioxide

d) Glutamate

e) Tetrahydrofolate

8- Which out of the following  conditions is associated with hypouricemia ?

a) Lesch Nyhan syndrome

b) Adenosine deaminase deficiency

c) Over activity of PRPP synthetase

d) Over activity of amido transferase

e) Von Gierke’s disease

9- In patients with Lesch Nyhan Syndrome, purine nucleotides are overproduced and over excreted. The hypoxanthine analogue Allopurinol, which effectively treats gout , has no effect on the severe neurological symptoms of Lesch- Nyhan patients because it does not-

a) Decrease de novo purine synthesis

b) Decrease de novo pyrimidine bio synthesis

c) Decrease urate synthesis

d) Increase PRPP levels (Phosphoribosyl pyrophosphate)

e) Inhibit xanthine oxidase

10- A 4- year old presents to a pediatric clinic with megaloblastic anemia and failure to thrive. Blood biochemistry reveals “Orotic aciduria”. Enzyme measurement of the white blood cells reveals a deficiency of pyrimidine biosynthesis enzyme Orotate Phospho ribose transferase and abnormally high activity of the enzyme Aspartate transcarbamoylase. Which of the following treatment will reverse all symptoms ?

a) Blood transfusion

b) Dietary supplementation of PRPP

c) Oral thymidine

d) Oral Uridine

e) Plasmaphresis

11)- Which of the following is a required substrate for purine biosynthesis ?

a) 5- methyl thymidine

b) Ara -C

c) Ribose phosphate

d) PRPP

e) 5-Fluoro uracil

12)- Which of the following is an analogue of hypoxanthine ?

a) Ara C

b) Allopurinol

c) Ribose phosphate

d) PRPP

e)5-FU

13)-A  Pentose with a 5′ phosphate group, a 2′ OH group and 1′ pyrimidine group describes which of the following structures ?

a) Cytosine

b) Thymidine

c) Thymidylate

d) Cytidylate

e) Guanosine

14) Which is the rate limiting step of pyrimidine synthesis that exhibits allosteric inhibition by cytidine triphosphate-

a) Aspartate transcarbamoylase

b) Hypoxanthine Guanine phosphoribosyl Transferase

c) Thymidylate synthase

d) Xanthine oxidase

e) PRPP synthetase

15)- The conversion of Inosine mono phosphate-

a) To Adenosine mono phosphate (AMP) is inhibited by Guanosine mono phosphate(GMP)

b) To AMP requires uridine mono phosphate (UMP)

c) To GMP requires GMP kinase

d) To GMP requires Glutamine

e) To Guanosine di phosphate (GDP) requires ribonucleotide reductase

16)- A 56-year -old diabetic with end stage renal disease receives a kidney transplant from his son. His nephrologist is concerned for the possibility of transplant rejection and puts the patient on mycophenolic acid, that inhibits which of the following enzyme in the synthesis of nucleotides ?

a) PRPP synthetase

b) IMP dehydrogenase

c) Adenylo succinate synthetase

d) Ribonucleotide reductase

e) Adenylosuccinase

17- A physician evaluates a 32-year-old patient for fatigue. The patient is found to have an elevated white blood cell count and an enlarged spleen. A referral to an oncologist results in a diagnosis of chronic myelogenous leukemia. Treatment with hydroxyurea, a ribonucleotide reductase inhibitor is begun. The normal functioning of this enzyme is to do which of the followings ?

a) Converts xanthine to uric acid

b) Converts ribonucleotides to deoxy ribonucleotides

c) Degrades guanine to xanthine

d) Degrades AMP to IMP

e) Converts PRPP to phosphoribosylamine

18- A child is noted to have recurrent respiratory infections that necessitate hospitalization. His lab tests demonstrate a decrease in T cells, B cells , natural killer cells and decreased antibodies. He is found to have severe combined immuno deficiency . The enzyme that is defective in this disorder is important in which of the following processes ?

a) Conversion of ribonucleotides to deoxy ribonucleotides

b) Formation of AMP

c) Synthesis of UMP

d) Conversion of dUMP to dTMP

e) Conversion of adenosine to inosine

19- A 7-year-old boy suffers from mental retardation and self-mutilation and has an increased levels of serum uric acid. These symptoms are characteristic of Lesch Nyhan syndrome, which is due  to defective-

a) Salvage pathway for pyrimidine biosynthesis

b) Denovo synthesis of pyrimidines

c) Xanthine oxidase

d) HGPRT (Hypoxanthine Guanine Phospho Ribosyl Transferase)

e) Formyl transferase

20- A 58-year-old man is awoken by a throbbing ach in his great toe. He had a similar attack earlier also, after indulging in a rich meal. On examination, he is noted to have an angry inflammed great toe and several nodules on the antihelix of his ear. Inhibition of which of the following enzymes might prevent the occurrence of such symptoms ?

a) Amido transferase

b) PRPP synthetase

c) Xanthine oxidase

d) Orotate phosphoribosyl transferase

e) Carbamoyl phosphate synthetase-II

 

Key to answers-  1)- c, 2)-d, 3)-c, 4)-c, 5)- b, 6)- c, 7)-a, 8)- b, 9)- a, 10)- a, 11)-d, 12)- b, 13)- d, 14)- a, 15)- d, 16)- b, 17)- b, 18)- e, 19)- d, 20)- c.

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1) Mechanism of action of cAMP

http://highered.mcgraw-hill.com/sites/0072507470/student_view0/chapter17/animation__second_messenger__camp.html

2) Steps of synthesis of pyrimidine nucleotides

http://www.wiley.com/college/fob/quiz/quiz22/22-5.html

3) Regulation of pyrimidine biosynthesis

http://www.wiley.com/college/fob/quiz/quiz22/22-7.html

 

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ATP

The nucleotide coenzyme adenosine triphosphate (ATP) is the most important form of chemical energy in all cells. All fuel sources of Nature, all foodstuffs of living things, produce ATP, which in turn powers virtually every activity of the cell and organism.  Cleavage of ATP is strongly exergonic. The energy  provided is used to drive endergonic processes (such as biosynthesis, movement and transport processes) through energetic coupling. ATP is the “most widely distributed high-energy compound within the human body”. The other nucleoside triphosphate coenzymes (GTP, CTP, and UTP) have similar chemical properties to ATP,but they are used for different tasks in metabolism.

Structure of ATP- In ATP, a chain of three phosphate residues are linked to the 5′-OH group of the nucleoside adenosine (see  figure-1).

ATP

 Figure-1- ATP is a nucleoside triphosphate containing adenine, ribose, and three phosphate groups. In its reactions in the cell, it functions as the Mg2+ complex

These phosphate residues are termed α, β, and γ. The α phosphate is bound to ribose by a phosphoric acid ester bond. The linkages between the three phosphate residues, on the other hand, involve much more unstable phosphoric acid anhydride bonds. The active coenzyme is in fact generally a complex of ATP with an Mg2+ ion, which is coordinatively bound to the β  and γ phosphates (Mg2+ ATP4–).

One phosphate ester bond and two phosphate anhydride bonds hold the three phosphates (PO4) and the ribose together. The construction also contains a β-N glycoside bond holding the ribose and the adenine together.

Energy of hydrolysis

Energy is usually liberated from the ATP molecule to do work in the cell by a reaction that removes one of the phosphate-oxygen groups, leaving adenosine diphosphate (ADP). When the ATP converts to ADP, the ATP is said to be spent. Then the ADP is usually immediately recycled in the mitochondria where it is recharged and comes out again as ATP.
11

Figure-2- showing the structure of ATP  (Adenosine tri phosphate). Adenosine attached to  two or one phosphate residues is called Adenosine di and mono phosphate respectively. The symbol ~ indicates that the group attached to the bond, on transfer to an appropriate acceptor, results in transfer of the larger quantity of free energy. For this reason, the term group transfer potential rather than “high-energy bond” is preferred . Thus, ATP contains two high-energy phosphate groups and ADP contains one, whereas the phosphate in AMP (adenosine monophosphate) is of the low-energy type, since it is a normal ester linkage.

In ATP, the oxygen atoms of all three phosphate residues have similarly strong negative charges . One of the reasons for the instability of phosphoric anhydride bonds is the repulsion between these negatively charged oxygen atoms, which is partly relieved by cleavage of a phosphate residue. In addition, the free phosphate anion formed by hydrolysis of ATP is better hydrated and more strongly resonance-stabilized than the corresponding residue in ATP. This also contributes to the strongly exergonic character of ATP hydrolysis.

Mechanisms of ATP formation

There are two basic mechanism involved for ATP formation-

1) Substrate level phosphorylation and  2) Oxidative phosphorylation

1) Substrate level phosphorylation- involves phosphorylation of ADP to form ATP at the expense of the energy of the parent substrate molecule without involving the electron transport chain.

Substrate is a high energy compound as compared to the product, the surplus energy is used for ATP formation.

Reactions of this type take place in glycolysis  and in the tricarboxylic acid cycle.

Examples-

a) Glycolysis

i) At the level of conversion of 1,3 BPG to 3, Phosphoglycerate

The reaction is catalyzed by phosphoglycerate kinase, phosphate is transferred from 1,3-bisphosphoglycerate onto ADP, forming ATP (substrate-level phosphorylation) and 3-phosphoglycerate. Since two molecules of triose phosphate are formed per molecule of glucose undergoing glycolysis, two molecules of ATP are formed at this stage per molecule of glucose undergoing glycolysis.

1

 Figure-3- Showing the conversion of 1,3 bisphosphoglycerate to 3, phosphoglycerate, and the formation of ATP by substrate level phosphorylation.

ii) At the level of conversion of phospho enol pyruvate to pyruvate

The phosphate of phosphoenolpyruvate is transferred to ADP by pyruvate kinase to form two molecules of ATP per molecule of glucose oxidized.

2

Figure-4- Showing the conversion of Phosphoenol pyruvate to pyruvate and second substrate level phosphorylation in Glycolysis.

 b) TCA cyclei) At the level of conversion of Succinyl co A to succinate by the enzyme succinate thiokinase (succinyl-CoA synthetase).

This is the only example in the citric acid cycle of substrate level phosphorylation. Tissues in which gluconeogenesis occurs (the liver and kidney) contain two isoenzymes of succinate thiokinase, one specific for GDP and the other for ADP. The GTP formed is used for the decarboxylation of oxaloacetate to phosphoenolpyruvate in gluconeogenesis. Nongluconeogenic tissues have only the isoenzyme that uses ADP.
3

Figure-5- Showing conversion of Succinyl co A to Succinate and formation of ATP by substrate level phosphorylation.

 c ) Another “energy-rich” phosphate compound is creatine phosphate, which is formed from ATP in muscle and can regenerate ATP as needed.

12

Figure-6- Showing the conversion of Creatine phosphate to creatine and formation of ATP by substrate level phosphorylation. 

2) Oxidative phosphorylation- Most cellular ATP does not arise in the way described above (i. e., by transfer of phosphate residues from organic molecules to ADP), but rather by oxidative phosphorylation.This process takes place in mitochondria (or as light-driven phosphorylation in chloroplasts) and is energetically coupled to a proton gradient over a membrane. These H+ gradients are established by electron transport chains and are used by the enzyme ATP synthase as a source of energy for direct linking of an inorganic phosphate to ADP. In contrast to substrate level phosphorylation, oxidative phosphorylation requires the presence of oxygen (i. e., aerobic conditions).

4

Figure-7- The flow of electrons guides the flow of protons across the innner mitochondrial membrane and that is coupled with release of energy which is captured for the phosphoryaltion of ADP to form ATP  in the electron transport chain

Functions of ATP

1) The ATP is used for many cell functions including transport work moving substances across cell membranes.

2) It is also used for mechanical work, supplying the energy needed for muscle contraction. It supplies energy not only to heart muscle (for blood circulation) and skeletal muscle (such as for gross body movement), but also to the chromosomes and flagella to enable them to carry out their many functions.

3) A major role of ATP is in chemical work, supplying the needed energy to synthesize the multi-thousands of types of macromolecules that the cell needs to exist.

4) ATP is also used as an on-off switch both to control chemical reactions.Phosphorylation of certain enzymes can increase or decrease their activities.

5) c AMP produced from ATP acts as second messenger for the hormonal action.

6) ATP allows the coupling of thermodynamically unfavorable reactions to favorable ones

Generally, ATP is connected to another reaction—a process called coupling which means the two reactions occur at the same time and at the same place, usually utilizing the same enzyme complex. Release of phosphate from ATP is exothermic (a reaction that gives off heat) and the reaction it is connected to is endothermic (requires energy input in order to occur). The terminal phosphate group is then transferred by hydrolysis to another compound, a process called phosphorylation, producing ADP, phosphate (Pi) and energy.

ATP can donate single phosphate, two phosphates or even Adenosine moiety to suitable acceptors for the formation of important biological compounds.

A) Single phosphate transfer

i) The phosphorylation of glucose to glucose 6-phosphate, the first reaction of glycolysis , is highly endergonic and cannot proceed under physiologic conditions.

ATP 2

To take place, the reaction must be coupled with another—more exergonic—reaction such as the hydrolysis of the terminal phosphate of ATP.

ATP3

When (1) and (2) are coupled in a reaction catalyzed by hexokinase, phosphorylation of glucose readily proceeds in a highly exergonic reaction that under physiologic conditions is irreversible. Many “activation” reactions follow this pattern.

ii) Phosphorylation of glycerol

 

8

Figure-8- Showing the phosphorylation of glycerol. Phosphorylation of glycerol is required for esterification to form trlacylglycerol, Glycerol kinase is absent in the adipose tissue.

B) Two phosphates transfer

i) Activation of fatty acids

During the process of activation of fatty acid before oxidation, ATP is converted to AMP with the release of pyrophosphate, which can subsequently be hydrolyzed to inorganic phosphates.

atp4

 Figure-9- showing the activation of fatty acid, a requirement for the complete oxidation of fatty acids in the mitochondria.

ii) Activation of amino acids

Amino acids are activated before incorporation into the growing peptide chain . The activation process can be represented as follows-

 

6

Figure-10- showing the activation of amino acid, a requirement for incorporation of amino acid in the growing peptide chain during translation.

AMP, formed as a consequence of several activating reactions involving ATP, is recovered by rephosphorylation to ADP.

Adenylyl Kinase (Myokinase) interconverts Adenine Nucleotides

This enzyme is present in most cells. It catalyzes the following reaction:

ATP5

Figure-11- showing the inter conversion of adenine nucleotides

This allows:

(1) High-energy phosphate in ADP to be used in the synthesis of ATP.

(2) AMP, formed as a consequence of several activating reactions involving ATP, to be recovered by rephosphorylation to ADP.

(3) AMP to increase in concentration when ATP becomes depleted and act as a metabolic (allosteric) signal to increase the rate of catabolic reactions, which in turn lead to the generation of more ATP .

C) Transfer of adenosine moiety

This takes place during activation of Methionine to S- Adenosyl Methionine (Active Methionine), which is a methyl group

donor in the body.
7

Figure-12- Showing the transfer of adenosine moiety to Methionine to form active Methionine (S-Adenosyl Methionine).

Why is ATP  considered the universal energy currency of cells why not other nucleotides like CTP, UTP etc ?

The other  nucleotides -GTP, CTP and UTP , do participate in metabolic reactions but ATP by virtue of the ease with which it can donate single phosphate, two phosphates, or even Adenosine moiety is considered a better nucleotide in energy transfer reactions . GTP has a role in gluconeogenesis and in the process of translation ; CTP is required for phospholipid and triacylglycerol synthesis , while UTP is required for glycogen synthesis and  also in Uronic pathway for the synthesis of  glycosaminoglycans and for detoxification reactions.

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Q.1- Give a brief description of functions of c- AMP ? Justify its role as a second messenger in hormonal action.

Q.2- Distinguish between:

a) Nucleoside and Nucleotide

b) Denaturation and Renaturation

c) Ribonucleotide and Deoxyribonucleotide

d) Uridine and Pseudouridine

e) Nucleotides in RNA and DNA

Q.3- Name the components of a nucleotide and show the order in which they are linked together,

Q.4-Why ATP is called the “energy currency of a cell”? Support your answer giving suitable examples.

Q.5- Name base and nucleoside analogs used as anticancer drugs.

Q,6- What is meant by hyperchromicity of denaturation?

Q.7- a) “Uracil is not present in DNA”, suggest the possible reason?

 b) ‘Thymine nucleotides are not present in RNA but exception to the rule is there’, give example in support of the statement.

Q.8- Discuss the biological significance of nucleotides?

Q.9- Compare and contrast the B and Z forms of DNA.

Q.10- Explain the extent to which the Watson-Crick structure of DNA is compatible with Chargaff’s rule.

Q-11- Explain how base-paired segments may occur in a single strand of RNA.

Q.12- Explain the following terms in the context of RNA structure: (a) poly A tail (b) cap.

Q.13- The following base sequence represents part of the transcribing strand of DNA     5’TACCATGGGCCC.3’

 (a) Give the orientation and base sequence of the complementary strand.

(b) Give the orientation and base sequence of the RNA that is  synthesized from it.

Q.14-Enlist the important differences between DNA and RNA

Q.15-Draw a well labeled diagram of secondary structure of t RNA and describe the significance of each of its arm. Why is t RNA called an adapter molecule?

Q.16- Discuss the functions of different types of RNAs present in a cell?

Q.17- Discuss the role played by Histones in DNA packaging?

Q.18- Draw a well labeled diagram showing the secondary structure of DNA. Discuss the salient features of Watson and Crick model of double stranded structure of DNA.

Q.19-Give a brief account of the small RNA s present in a cell. Discuss the significance of each of them.

Q.20-What is meant by polarity of DNA? What is its significance in replication or transcription mechanisms?

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