Home Assignment

1. 2. 3. 4. 5. 6. 7. a) b) c) d) e) 8. 9.

Home assignment (Vitamins and co-enzymes) What is the structural relationship between Vitamin D3 and Cholesterol? Why it is possible to argue that Vitamin D is not a vitamin? Give a reason for the toxicity caused by the overdose of the lipid-soluble of vitamins? Why can some vitamin-K antagonists act as anticoagulants? What is the role in vision of the cis-trans isomerization of retinal? What is unique about TPP that makes it useful in decarboxylation reaction? Choose the correct answers from below Tightly bounded coenzymes are often referred as (i) Cofactors, (ii) Prosthetic groups, (iii) Vitamins, (iv) Metaloenzymes. Which vitamin is the precursor of acyl carrier protein CoASH (i) Vitamin B1, (ii) Vitamin B6, (iii) Vitamin B5, (iv) Vitamin B12. In the initial process of vision retinal binds with which of the following protein (i) Opsin, (ii) Rhodopsin, (iii) Bathorhodopsin, (iv) Metarhodopsin II. The main role of Vitamin K in blood coagulation is (i) Generation of fibrin multimer, (ii) Generation of Prothrombin, (iii) Activation of Fibrinogen, (iv) Formation of Thrombin. Biotin binds strongly with which of the following protein (i) Hemoglobin, (ii) Pyruvate decarboxylase, (iii) Streptavidin, (iv) Thermolysin. When 14C-labeled 4-hydroxyproline was administered to rats, the 4hydroxyproline in newly synthesized collagen was not radiolabeled. Explain? Draw the mechanism of transamination reaction with pyridoxal phosphate.

Home Assignment (Protein isolation & purification) 1. Sephadex G-75 has an exclusion limit of 80KD. If you tried to use this column material to separate alcohol dehydrogenase (150 KD) from β-amylase (200KD), what would happen? 2. Referring to the question above, could you separate β-amylase from bovine serum albumin (66KD) using this column? 3. What could be an advantage of using an anion exchange column based on quaternary amines (i.e, resin-N+Et3) as opposed to a tertiary amine (resinNH+Et2)? 4. Gel-Filtration chromatography is a useful method for removing salts, such as ammonium sulphate, from protein solutions. Describe how such separation is accomplished? 5. Why is the order of separation based on size opposite for gel-filtration and gelelectrophoresis, even though they often use the same compound to form the matrix? 6. Design an experiment to purify protein X on an anion-exchanger column. Protein X has an isoelectric point of 7.0. 7. A Gel Chromatography column of Bio-Gel- P 30 with a bed volume of 100mL is poured. The elution volume of the protein hexokinase (96KD) on this column is 34mL. That of an unknown protein is 50mL. What are the void volume of the column, the volume occupied by the gel and the relative elution volume of the unknown protein? What would be the rough molecular weight of the unknown protein? 8. How can gel-filtration chromatography be used to arrive at an estimate of the molecular weight of a protein? 9. Why do most people elute bound proteins from an ion-exchange column by raising the salt concentration instead of changing the pH?

Protein sequencing 1. Why can the Edman degradation not be used effectively with very long peptides? 2. What should happen during an amino acid sequencing experiment using the Edman degradation if you accidentally added twice as much Edman reagent as the peptide you were sequencing? 3. A sample of an unknown protein was divided into two aliquots. One aliquot was treated with trypsin, and the other with CNBr. Given the following sequences (N to C) of the resulting fragments, deduce the sequence of original peptide. Trypsin treatment Asn-Thr-Trp-Met-Ile-Lys Gly-Tyr-Met-Gln-Phe Val-Leu-Gly-Met-Ser-Arg CNBr Treatment Gln-Phe Val-Leu-Gly-Met Ile-Lys-Gly-Tyr-Met Ser-Arg-Asn-Thr-Trp-Met 4. A sample of peptide of unknown sequence was treated with trypsin; another sample of same peptide was treated with chymotrypsin. The sequences (N to C) of the smaller peptides are Trypsin digestion Met-Val-Ser-Thr-Lys Val-Ile-Trp-Thr-Leu-Met-Ile Leu-Phe-Asn-Glu-Ser-Arg Chymotrypsin digestion Asn-Glu-Ser-Arg-Val-Ile-Trp Thr-Leu-Met-Ile Met-Val-Ser-Thr-Lys-Leu-Phe Deduce the complete sequence of that peptide. 5. A polypeptide is subjected to the following degradative techniques resulting in polypeptide fragments with the indicated amino acid sequences. What is the complete sequence of that peptide? CNBr Treatment Asp-Ile-Lys-Gln-Met Lys Lys-Phe-Ala-Met Tyr-Arg-Gly-Met Trypsin digestion

Gln-Met-Lys Gly-Met-Asp-Ile-Lys Phe-Ala-Met-Lys Tyr-Arg 6. While on an expedition to the Amazon jungle, you isolate a polypeptide you suspect of being the growth hormone of a giant spider. Unfortunately, your portable sequencer was so roughly treated that it refuses to provide the sequence of not more than four consecutive amino acid residues. Nevertheless, you preserve and obtain the following data: Hydrazinolysis Val Dansyl chloride treatment followed by acid hydrolysis Dansyl-Pro Trypsin treatment Gly-Lys Phe-Ile-Val Pro-Gly-Ala-Arg Ser-Arg Provide as much information you can get from the above results to predict its complete sequence. 7. Determine the sequence of hexapeptide on the basis of the following data. N-terminal analysis: A Trypsin digestion: R,A,V and R,S,Y Carboxypeptidase digestion: No digestion Chymotrypsin digestion: A,R,V,Y and R,S 8. Determine the sequence of a peptide consisting of 14 amino acids on the basis of the following data N terminal analysis: S Carboxypeptidase Digestion: L Trypsin digestion: 3S, 2L, F, I, M, T, W and G, K, S, Y Chymotrypsin digestion: F, I, S and G,K,L and L,S and M,T and S,W and S,Y CNBr treatment: 2S, F,G,I,K,L,M,T,Y and 2S,L,W Enzyme catalysis 1. The following reaction is catalyzed by an enzyme at pH 7.0 at RT, whereas nonenzymatically, this reaction does not occur under these conditions. Explain how the enzyme can easily catalyze this reaction. O -H2O

O

OH +H2O

2. Other things being equal, what is a potential disadvantage of an enzyme having a very high affinity for its substrate? 3. Amino acids that are far apart in the amino acid sequence of an enzyme can be essential for its catalytic activity, what does this suggest about its active site.

4. If only a few amino acid residues of an enzyme are involved in its catalytic activity, why does the enzyme need such a large number of amino acids? 5. Indicate what interactions are relevant by each letter in the following picture?

6. In the following reaction the formation of two products has been detected, elucidate the possible mechanisms? O F

D O

D

Glyoxylase F

O

O OH

+

OH

SG O

SG: Glutathione

7. α-chloromethyl ketones are acting as chymotrypsin inactivator. When (2S)-Nacetyl-L-alanyl-L-phenylalanyl-α-chloroethane is treated with γ-chymotrypsin, its activity was inhibited by the action of the chloroketone compound. The crystal structure of the covalent adduct with the enzyme shows that the stereochemistry of the inactivator is retained. Based on this information elucidate a detail mechanism of the enzyme inactivation?

O H N

AcNH O

Me H Cl Ph

(2S)-N-acetyl-L-alanyl-L-phenylalanyl-α-chloroethane

10. Explain why mutating all three residues of trypsin’s catalytic triad has essentially no greater effect on the enzymes catalytic rate enhancement than mutating only Ser 195. 9. Explain why γ-pyridone is not nearly as effective a catalyst for glucose mutarotation as is α-pyridone. What about β-pyridone?

Protein Structure 1. What is a reverse turn? Draw two types of reverse turns. 2. Why proline is frequently encountered at the places in the myoglobin and hemoglobin molecules where the polypeptide chain turns a corner? 3. Woolen clothing shrinks when washed in hot water, but items made of silk do not, suggest a reason? 4. List some of the differences between the α-helix and β-sheet forms of a secondary structure? 5. List five forces that are responsible for maintaining the correct three-dimensional shapes of proteins. Specify which groups on the protein are involved in each type of interaction? 6.