1.11 ANSWER TO CRITICAL THINKING QUESTIONS

Structure of atoms, molecules and chemical bonds 1. What is the approximate percentage (in mass) of water in the human body? Is this percentage expected to be larger in the adult or in the old individual?

Ans) Approximately 65% of the human individual mass is water. The brain, for example, has around 90% of water in mass, the muscles, 85%, and the bones have between 25% and 40% of water. Younger adult individuals have proportionally more water in mass than older individuals.

2. Can the heat capacity of water be considered small or large? What is the biological significance of that characteristic?

Ans) Water has a specific heat of 1 cal/g.oC which means that 1 oC per gram is changed in its temperature with the addition o r subtraction of 1 cal of energy. This is a very elevated value (for example, the specific heat of ethanol is 0.58 cal/g °C, and mercury, a metal, has a specific heat of 0.033 cal/g. °C) making water an excellent thermal protector against variations of temperature. Even if sudden external te mperature changes occur, the internal biological conditions are kept stable in organisms which contain enough water.

3. Which of the three species CO3 2-, CO2 and CO has the shortest C-O bond length? Explain the reason for your answer.

Ans) CO has the shortest bond because there is a triple bond, or because the bond order is 3, meaning it has the greatest number of shared electrons between the carbon and the oxygen atom.

4. Ammonia, NH3, is very soluble in water, whereas phosphine, PH3, is only moderately soluble.

Ans) Ammonia has hydrogen-bonding intermolecular forces, whereas phosphine has dipole- dipole and/or dispersion intermolecular forces.

Water also has hydrogen-bonding intermolecular attractive forces. Ammonia is more soluble in water than phosphine because ammonia molecules can hydrogen-bond with water molecules, whereas phosphine molecules cannot hydrogen-bond with water molecules.

5. Which of the following describes the changes in forces of attraction that occur as H2O changes phase from a liquid to a vapor? A. H-O bonds break B. Hydrogen bonds break C. Ionic bonds break D. Covalent bonds become more effective.

Ans) B

Composition, structure and function of biomolecules (carbohydrates, lipids, proteins, nucleic acids and vitamins) 1. Why is glycine a highly conserved amino acid residue in the evolution of proteins?

Ans. Glycine has the smallest side chain of any amino acid. Its smallness is critical in allowing polypeptide chains to make tight turns or to approach one another closely.

2. The gene encoding a protein with a single disulphide bond undergoes a mutation that changes a serine residue into a cysteine residue. Propose a direct method to find out whether the disulphide pairing in this mutant is the same as in the original protein.

Ans. The positions of the disulphide bond can be determined by diagonal electrophoresis. The disulphide pairing is unaltered by the mutation if teh off-diagonal peptides formed fromt eh native and mutant proteins are the same.

3. The atmosphere of the primitive earth before the emergence of life contained N2, NH3, H2, HCN, CO and H2O. Which of these compounds is the most likely precursor of most of the atoms in adenine? Why?

Ans. HCN. Adenine can be viewed as a pentamer of HCN.

4. Are organic solvents like benzene and ether polar or non-polar substances?

Ans) Benzene and the ethers are molecules without electrically charged portions and thus they are non -polar substances.

5. How to distinguish between glucose and sucrose?

Ans) Glucose is reducing sugar, it reacts with tollens reagent to form silver mirror, while sucrose is a non-reducing sugar. It does not react.

Stabilizing interactions (Van der Waals, electrostatic, hydrogen bonding, hydrophobic interaction, etc.) 1. Why does Poly-L-leucine, in an organic solvent like dioxane, is alpha-helical whereas poly-L-isoleucine is not?

Ans. The methyl group attached to the beta carbon of isoleucine sterically interferes with alpha helix formation. In leucine this methyl group is attached to the gamma carbon atom, which is farther from the main chain and hence does not interfere.

2. What type of non-covalent interactions holds together Graphite?

Ans) van der Waals Interactions

3. A mutation that changes Ala to Val in the interior of a protein leads to loss of activity. A 2nd mutation in different position changes Isoleucine to glycine. How does this restore the activity?

Ans) The first mutation destroys activity because valine occupies more space than alanine does, so protein must take a differ ent shape, assuming this residue lies in the closely packed interior. The second mutation restores activity because of compensatory reduction in volume; glycine is smaller than isoleucine.

4. Why is it not correct to assert that DNA self-replicates?

Ans) DNA is not completely autonomous in its duplication process because the replication do es not occur without enzymatic activity. So it is not entirely correct to assert that DNA self-replicates.

5. Do the phosphate and the pentose groups give homogeneity or heterogeneity to the nucleic acid chains? What about the nitrogen-containing groups?

Ans) The phosphate and the pentose groups are the same in every nucleotide that forms the nucleic acid and so they give homog eneity to the molecule. The nitrogen-containing bases however can vary among adenine, thymine, cytosine, guanine (in DNA) and uraci l (in RNA). These variations provide the heterogeneity of the nucleic acid molecule.

Principles of biophysical chemistry (pH, buffer, reaction kinetics, thermodynamics, colligative properties) 1. What is the 'pH' of pure water and that of rain water? Explain the difference.

The pH of pure water is seven. Rain water is slightly acidic because as rain drop fall, the carbon dioxide in the air dissolv es with drops to form very weak carbonic acid. Accordingly, rain water has a pH that is slightly below 7.

2. What is the pH of solution 'A' which liberates CO2 gas with a carbonate salt? Give the reason?

The pH of solution 'A' is lesser than 7. Carbonates salts react with acids (A) to liberate CO2 gas.

3. What is a universal indicator? What is its advantage?

A universal indicator is a mixed indicator of organic chemicals which not only shows whether the given solution is acidic or basic, but also shows the approximate pH values by giving a wide particular colour for a specific value of pH.

4. Why can't hydrogen ions exist by themselves?

Ans) It is possible to have isolated H+ ions in the gas phase. But if a free hydrogen ion encounters a water molecule, it att acks the unshared electron pairs on the oxygen in the water molecule and forms a hydronium ion, H3O+. The chemical bond that forms between the water and hydrogen ion is covalent and very strong. In an aqueous solution, essentially all of the H+ exists as H3O+ .

5. Is a negative pH possible?

Ans) It's possible. If the molarity of hydrogen ions is greater than 1, you'll have a neg ative value of pH. For example, you might expect a 12 M

HCl solution to have a pH of -log(12) = -1.08. There are some complications in high molarity acid solutions that make pH calculations from acid molarity inaccurate and difficult to verify experimentally.

Bioenergetics, glycolysis, oxidative phosphorylation, coupled reaction, group transfer, biological energy transducers 1. Suppose that you wound a spring tightly, clamped it so that it could not unwind, and then placed the spring in acid, dissolving it without unwinding. What would happen to the energy stored in the spring?

Ans) The solution becomes Warm.

2. Suppose that you determined ΔG for a reaction at room temperature and then carried out the same reaction at a higher temperature. At higher temperatures, what will the change in ΔG for the reaction as compared to that at lower temperatures?

Ans) A larger negative number than at room temperature.

3. The first step in glycolysis is phosphorylation of glucose to form glucose-6-phosphate. What is the importance of this step?

Ans) To make glucose more polar, locking it within the cell.

4. If we can convert glucose to pyruvic acid and to other metabolites, we should be able to simply reverse glycolysis and form new glucose from pyruvic acid. What prevents this?

Ans) The free energy changes for some of the reactions that lead from glucose to pyruvate are too large and negative for easy reversal.

5. Oligomycin Inhibits synthesis of ATP during oxidative phosphorylation. What is the mechanism of inhibition?

Ans) Oligomycin inhibits mitochondrial ATPase and thus prevents phosphorylation of ADP to ATP. It prevents utilization of ene rgy derived from electron transport for the synthesis of ATP. Oligomycin has no effect on coupling but blocks mitochondrial phosphorylation so that bot h oxidation and phosphorylation cease in its presence.

Principles of catalysis, enzymes and enzyme kinetics, enzyme regulation, mechanism of enzyme catalysis, isozymes 1. Suppose a mutant enzyme binds a substrate 100-fold as tightly as does the native enzyme. What is the effect of this mutation on the catalytic rate if the binding of the transition state is still unaffected?

Ans. The mutation slows the reaction by a factor of 100 because the activation free energy is increased by +2.72 kcal/mol (2.303RTlog100).

Strong binding of the substrate relative to the transition state slows catalysis.

2. What is a simple means of determining whether a recently discovered proteolytic enzyme is a thiol protease?

Ans. The essential sulfhydryl groups in its active site should be highly susceptible to chemical modification. The enzyme sh ould be inactivated by reacting it with iodoacetamide, N-ethylmaleimide or any other sulfhydryl-specific reagent. Bound sulfhydryl is likely to protect the key sulfhydryl from alkylation.

3. The HIV 1 protease, like other retroviral proteases, is a dimer of identical subunits rather than a single chain twice as large. What is the selective advantage to the virus of a dimeric arrangement?

Ans. Less RNA is needed to encode the protease. Small genome is more readily replicated and packaged than a large one. Also, the protease does not become active until a critical concentration is attained. Premature excision of the viral subunits from the polyprotein is thereby prevented.

Unit 1

4. You have isolated a dimeric enzyme that contains 2 identical active sites. The binding of substrate to one active site decreases the substrate affinity of the other active site. Which allosteric model best accounts for this negative cooperativity?

Ans. The sequential model can account for the negative cooperativity, whereas the concerted model cannot. Homotropic alloste ric interactions should be cooperative if concerted model holds.

5. Antithrombin III forms an irreversible complex with thrombin but not with prothrombin. What is the most likely reason for this difference in reactivity?

Ans. Antithrombin III is a very slowly hydrolyzed substrate of thrombin. Hence its interaction with thrombin requires a fully formed active site on the enzyme.

Conformation of proteins (Ramachandran plot, secondary structure, domains, motif and folds) 1. How can curls be induced in hair?

Ans. Disulphide bonds in hair are broken by adding a thiol and applying gentle heat. The hair is curled and an oxidising agent is added to re form the disulphide bonds and stabilize the desired shape.

2. Suppose that a protease is synthesized by the solid-phase method from D rather than L amino acids. How would the sedimentation, electrophoretic and circular dichroism properties of this enzyme compare with those of the native form?

Ans. The sedimentation and electrophoretic properties of the L-enzyme and the mirror image D form would be the same. The circular dichroism spectra would have the same magnitude but be of opposite sign because the two structures have opposite screw sense.

3. Proteins that span biological membranes often contain alpha helices. Why is an alpha-helix particularly suited to exist in a hydrophobic environment?

Ans) The amino acids in the alpha helix would be hydrophobic in nature. All the amide hydrogen atoms and carbonyl oxygen atom s of the peptide backbone take part in intrachain H bonds, thus stabilizing these polar atoms in a hydrophobi c environment.

4. What is the essential condition for a protein to be identical to another protein?

Ans) For a protein to be identical to another protein it is necessary for the sequence of amino acids that form them to be id entical.

5. In sickle cell anemia, are all of the structural levels of the protein modified?

Ans) In sickle cell disease there is a change in the primary protein structure of one of the polypeptide chains that form hem oglobin: the amino acid glutamic acid is substituted by the amino acid valine in the β chain. The spatial conformation of the molecule in addition is also affected and modified by this primary “mistake” and the modification also creates a different (sickle) shape to the red blood cells. Modified, sickled, red blood cells sometimes aggregate and obstruct the peripheral circulation causing tissue hypoxia and the pain crisis typical of sickle cell anemia.

Conformation of nucleic acids (helix (A, B, Z), t-RNA, micro-RNA) 1. How does cordycepin (3’-deoxyadenosine) block the synthesis of RNA?

Ans. Cordycepin terminates RNA synthesis. An RNA chain containing cordycepin lacks a 3’ OH group.

2. A negatively supercoiled DNA molecule undergoes a B to Z transition over a segment of 360 base pairs. What is the effect on the writhing (supercoiling)?

Ans) +66

The twist changes from that in B-form (TB) to that in Z DNA (TZ):

TB = 360/ +10 = +36 and TZ = 360/-12 = -30

ΔT = TZ - TB = - 30 - (+36) = -66

ΔT = TZ - TB = - 30 - (+36) = -66

ΔL = 0

ΔW = -ΔT = -(-66) = +66

3. Why is HAP column used to distinguish single stranded and double stranded DNA?

Ans) HAP (hydroxyapatite) column. Duplex nucleic acids will bind to HAP at room temperature, whereas single -stranded nucleic acids will elute.

The duplex fraction can subsequently be retrieved from the column by heating it, melting the nucleic acid and now collecting it as it elutes.

4. To precipitate DNA, an alcohol like ethanol or propanol is added to an aqueous DNA solution. Why should Na+ or NH4+ also be added?

Ans) a counter ion (Na+ or NH4+) must be present in order for the negatively-charged DNA to form a salt and precipitate.

5. A 41.5 nm-long duplex DNA molecule in the B-conformation adopts the A conformation upon dehydration How long is it now? What is its approximate number of base pairs?

Ans) 27.3 nm and 122 bps

For B-DNA, 41.5 nm has 41.5/0.34 = 122 bps that make 122/10 = 12.2 turns.

If converted to A-DNA, these 122 base pairs will now make: 122/11 = 11.1 turns with an overall length of 122 * 0.224 = 27.3nm

Stability of proteins and nucleic acids 1. Suppose you want to radioactively label DNA but not RNA in dividing and growing bacterial cells. Which radioactive molecule would you add to the culture medium?

Ans) Tritiated thymine or tritiated thymidine

Unit 1

2. RNA is readily hydrolyzed by alkali whereas DNA is not. Why?

Ans) The 2’ OH group in RNA acts as an intramolecular nucleophile. In the alkaline hydrolysis of RNA, it forms a 2’-3’ cyclic intermediate.

3. The amino acid coded by codon GGG could not be deciphered in the same way as UUU, CCC or AAA. Why is p olyG an ineffective template?

Ans) PolyG forms a triple helical structure. Only single stranded RNA can serve as a template for protein synthesis.

4. Griffith used heat-killed S. Pneumococci to transform R mutants. Studies years later showed that dsDNA is needed for efficient transformation and that high temperature melt the DNA double helix. Why were Griffith’s experiments nevertheless successful?

Ans. The DNA renatured when the heat killed pneumococci were cooled before they were injected into mice.

5. In designing of primers, the Tm of each primer should approximately be the same. What is the basis of this requirement?

Ans) If the Tm of the primers are too different, the extent of hybridization with the target DNA will differ during the annea ling phase, which would result in differential replication of the strands.

Metabolism of carbohydrates, lipids, amino acids nucleotides and vitamins 1. When we digest food and use it to produce energy, we convert a few large, complex molecules into many small, simple molecules. Under these circumstances, what is the effect on entropy?

Ans) Entropy increases.

2. In addition to the regulators of enzyme activity within the citric acid cycle, two enzymes outside the cycle profoundly affect its activity. Which ones?

Ans) Pyruvate carboxylase and pyruvate dehydrogenase.

3. If you were handed a sample of a white, greasy substance and asked to determine whether it were a triacylglycerol or a fatty acid, how would you do it?

Ans) By mixing it with base to see whether it dissolved. Fatty acids readily react with bases to form soap solutions.

4. Why are Arachidonic acid and EPA are classified as essential?

Ans) They are needed for synthesis of eicosanoids.

5. Fatty acid oxidation occurs mostly within mitochondria, but fatty acids themselves cannot easily cross the mitochondrial membrane. How do they pass?

Ans) As esters of carnitine.