Home assignment (Vitamins and co-enzymes)

1. What is the structural relationship between Vitamin D3 and Cholesterol?

2. Why it is possible to argue that Vitamin D is not a vitamin?
3. Give a reason for the toxicity caused by the overdose of the lipid-soluble of
vitamins?
4. Why can some vitamin-K antagonists act as anticoagulants?
5. What is the role in vision of the cis-trans isomerization of retinal?
6. What is unique about TPP that makes it useful in decarboxylation reaction?
7. Choose the correct answers from below
a) Tightly bounded coenzymes are often referred as (i) Cofactors, (ii) Prosthetic
groups, (iii) Vitamins, (iv) Metaloenzymes.
b) Which vitamin is the precursor of acyl carrier protein CoASH
(i) Vitamin B1, (ii) Vitamin B6, (iii) Vitamin B5, (iv) Vitamin B12.
c) In the initial process of vision retinal binds with which of the following protein
(i) Opsin, (ii) Rhodopsin, (iii) Bathorhodopsin, (iv) Metarhodopsin II.
d) 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.
e) Biotin binds strongly with which of the following protein (i) Hemoglobin, (ii)
Pyruvate decarboxylase, (iii) Streptavidin, (iv) Thermolysin.
8. When 14C-labeled 4-hydroxyproline was administered to rats, the 4-
hydroxyproline in newly synthesized collagen was not radiolabeled. Explain?
9. 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 (resin-
NH+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 gel-
electrophoresis, 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 O
-H2O
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 O O
Glyoxylase D
F D F OH + SG
O OH O

SG: Glutathione
7. α-chloromethyl ketones are acting as chymotrypsin inactivator. When (2S)-N-
acetyl-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 Me
N
AcNH H
O 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.