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Mechanism

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									                                          University of Cape Town
                                    Department of Molecular & Cell Biology

Course Code:    MCB2014F
Course Name: Macromolecules                           Test 1                                       April 2008

(Total = 65 marks)
Student Number

A.       Water and pH (5 marks)

The following questions (1 to 5) are of the multiple choice type (choose the most appropriate answer):

1.   Lemon juice at pH 2.2 contains about _____ times as much H+ as orange juice at pH 4.3.
         a.      10-2
         b.      0.9
         c.      2.1
         d.      21
         e.      126                                                                                        (1)

2.   If equal amounts of NaH2PO4 and Na2HPO4 are mixed in water, calculate the resulting pH. The pKas of
     phosphoric acid are 2.1, 7.2, and 12.4.
           a.     2.1
           b.     4.5
           c.     7.2
           d.     9.8
           e.     12.4                                                                                   (1)

3.   Which of the following pairs would be the best buffer at pH 10.0?
         a.       Acetic acid and sodium acetate (pKa = 4.76)
         b.       H2CO3 and NaHCO3 (pKas are 3.77 and 10.4)
         c.       Lactic acid and sodium lactate (pKa = 3.86)
         d.       NaH2PO4 and Na2HPO4 (pKas are 2.1, 7.2, 12.4)
         e.       Sodium succinate and succinic acid (pKa = 4.21)                                           (1)

4.   Buffers have all of the following characteristics EXCEPT:
           a.     they have relatively flat titration curves at the pH(s) where they buffer.
           b.     they resist changes in their pH as acid or base is added.
           c.     they are typically composed of a weak acid and its conjugate base.
           d.     they buffer best for polyprotic acids half-way between the two pKa values.
           e.     buffer where the amounts of conjugate base are nearly equivalent to the amounts of weak acid.
                                                                                                            (1)
5.   When preparing an acetate buffer at pH 4.5 with 0.01 M solutions of acetic acid (pK a = 4.8) and sodium acetate,
     the volume of acetic acid needed would be _________________ the volume of sodium acetate solution.
           a.     equal to
           b.     less than half of
           c.     more than half of
           d.     about six times
           e.     about twice                                                                               (1)

B.       Thermodynamics of Biological Systems (5 marks)

The following questions (1 to 5) are of the multiple choice type (choose the most appropriate answer):

1.   Calorimetry measures ______________ by a biochemical process
           a.    heat absorbed or given off, H,
           b.    pressure change inside calorimeter created
           c.    water pressure created
           d.    entropy change, S,
           e.    volume change, V, created                                                                 (1)

                                                                                                                  1
2.   Which equation defines a system at equilibrium?
         a.      G > 0
         b.      G° = G
         c.      G = 0
         d.      G° = 0
         e.      G = RT ln ([products]/[reactants])                                                    (1)

3.   Thermodynamic parameters (entropy, enthalpy, free energy, and internal energy) are given for an unknown
     enzyme. Explain which results would be expected for the breaking of hydrogen bonds and the exposure of
     hydrophobic groups from the interior during the unfolding process of a protein.
          a.     Entropy change, S, is zero
          b.     Entropy change, S, is negative
          c.     The reaction is spontaneous
          d.     Enthalpy change, H, is negative
          e.     Enthalpy change, H, is positive                                                (1)

4.   "High-energy" compounds exhibit large negative free energy of hydrolysis and include all EXCEPT:
          a.     phosphate esters.
          b.     enol phosphates and phosphate anhydrides.
          c.     acyl phosphates.
          d.     guanidino phosphates.
          e.     thioesters.                                                                    (1)

5.   The chemical reasons for the large negative Go' for the hydrolysis of ______ include destabilization of the
     reactant due to bond strain caused by electrostatic repulsion, stabilization of the products by ionization and
     resonance, and entropy factors due to hydrolysis and subsequent ionization.
           a.      AMP
           b.      ATP
           c.      PEP (phosphoenolpyruvate)
           d.      Phosphocreatine
           e.      All of the above                                                                    (1)

C.       Amino acids (25 marks)

1.   An idealized titration curve of 1 mol of a fully protonated amino acid titrated with sodium hydroxide is shown
     in the following figure (13 marks):




     (a) What is the name of the amino acid that is being titrated?        ANS:…………………                         (1)
     (b) Draw the structures (and reactions) for the three ionizations of this protonated amino acid.          (4)



                                                                                                                      2
     (c) What regions in the graph correspond to each ionization step (choose from I to VII)?
        ANS:……………………                                                                                          (3)
     (d) At what pH value is the average nett charge on the amino acid equal to +1 (read off the graph)?
        ANS:………………………..                                                                                       (1)
     (e) At what pH value is the average nett charge on the amino acid equal to + ½ (read off the graph)?
        ANS:……………………..                                                                                        (1)
     (f) At what pH value is the average nett charge on the amino acid equal to -1 (read off the graph)?
          ANS:…………………..                                                                                       (1)
     (g) What point corresponds to the isoelectric point of this amino acid (choose from I to VII)?
        ANS:……………..                                                                                           (1)
     (h) At which point is the pH equal to the pKa value for the dissociation of the carboxyl group?
          ANS:………………..                                                                                        (1)

2.   Only one of the 20 common proteinaceous amino acids has no free -amino group.
         (a) Name this amino acid.                                                                            (1)
     ANSWER:…………………….
         (b)      Draw the structure of the amino acid mentioned in (a)                                       (1)
         ANSWER:




         (c) Where in a protein is this amino acid commonly found? (1)
         ANSWER:…………………………………………………………………………………………………
         ……………………………………………………………………………………………………………

3.   Draw a simple mechanism for the reaction of a cysteine sulfhydryl group with iodoacetamide.              (3)
     ANSWER:




The following questions (4 to 9) are of the multiple choice type (choose the most appropriate answer):

4.   The pKa of the cysteine side chain sulfhydryl group is 8.3, so it is about 77% dissociated at pH ______.
         a. 8.0
         b. 8.2
         c. 8.4
         d. 8.6
         e. 8.8                                                                                               (1)

5.   All of the statements about the classification of these amino acids are correct EXCEPT:
          a. Glutamine and Asparagine are polar, uncharged amino acids.
          b. Alanine and valine are neutral, nonpolar amino acids.
          c. Threonine and glycine are polar, uncharged amino acids.
          d. Lysine and histidine are basic amino acids.
          e. Methionine and cysteine are aromatic amino acids.                                                (1)

6.   Which of the following amino acids has more than one chiral carbon?
        a. serine
        b. isoleucine
        c. methionine
        d. cysteine
        e. aspartic acid                                                                                      (1)




                                                                                                                    3
7.   The pKa of the -COOH group is ___________ by the presence of the -NH3+ group on an amino acid.
         a.       greatly decreased (> 2 pH units)
         b.       greatly increased (>2 pH units)
         c.       unchanged
         d.       slightly decreased (~ 1.5 pH units)
         e.       slightly increased (~1.5 pH units)                                                 (1)

8.   Which statement is INCORRECT about these amino acids, and amino acid derivatives?
        a. Thyroxine is a phosphorylated amino acid that is found only in thyroglobulin in the thyroid gland.
        b. Ornithine is an important metabolic intermediate.
        c. Epinephrine is a hormone derived from tyrosine.
        d. Serotonin is a neurotransmitter derived from tryptophan.
        e. Histamine is a neurotransmitter derived from histidine.                                        (1)

9.   The amino acid composition of a protein gives:
         a. the sequence of the protein.
         b. the number of residues of each amino acid in the protein.
         c. the molecular weight of the protein.
         d. the percentage or ratio of the various amino acids in the protein.
         e. an identification of the N-terminal and C-terminal amino acids.                                                                                       (1)

D.         Proteins (30 marks)

1.   Complete this purification table: fill-in the correct values for each column and then use the completed table to
     answer the following questions. Explain your answers with appropriate calculations (12 marks).

                           Protein                  Enzyme                     Specific                   Yield %                   Purification
                           (mg)                     activity                   activity                                             factor (fold
                                                    (units)                    (units/mg)                                           purification)
Crude extract              1.00 x 104               5.00 x 103                 0.5                        100                       1
(NH4)2SO4                  4.00 x 103               4.00 x 103
Gel filtration             2.00 x 102               3.00 x 103
Ion-exchange               0.80 x 102               1.90 x 103
Affinity column            0.50 x 10                1.60 x 103
                                                                                                                                                                  (6)
(a) Which single chromatographic step had the highest purification (fold purification)?                                                                           (1)
     ANSWER:………………………….
(b) Which single chromatographic step had the lowest purification?                                     (1)
    ANSWER:…………………………..
(c) Which chromatographic step would you consider excluding from future attempts to purify the mystery enzyme?
                                                                                                                                                                  (1)
     ANSWER:………………….
(d) Briefly describe the principle of ammonium sulfate precipitation in separating out proteins.                                                                  (3)
     ANSWER:………………………………………………………………………………………………………....
     ..............……………………………………………………………………………………………………………
     ……………..................................................................................................................................................................
     …………………………………………………………………………………………………………………….
2.   Briefly explain why is silk fibroin so strong, but at the same time so soft and flexible                                                                     (2)
     ANSWER:………………………………………………………………………………………………………
     ……………………………………………………………………………………………………………………
     ………………………………………………………………………………………………….........................
     ………………………………………………………………………………………………………………….


                                                                                                                                                                         4
3.   In the protein adenylate kinase, the C-terminal region is -helical, with the following sequence:
     ---Val-Asp-Asp-Leu-Phe-Ser-Gln-Leu-Cys-Thr-His-Leu-Asp-Thr-Leu-Lys---
     The hydrophobic residues in this sequence are presented in boldface type. Suggest a possible reason for the
     periodicity in their spacing.                                                                          (2)
     ANSWER:………………………………………………………………………………………………………
     ………………………………………………………………………………………………………………….


4.   Polyglycine, a simple polypeptide, can form a helix with  = -80°,  = +150°. From the Ramachandran plot
     (see figure below), describe this helix with respect to:
     (a) handedness                                                                                         (1)
     ANSWER:……………….
     (b) number of residues per turn.                                                                       (1)
     ANSWER:………………..




5.   Write down the  and  values for a normal right-handed -helix.                                       (2)
     ANSWER:
                  = …………..
                  = ………….

6.   What are the structural and functional advantages driving quaternary association, or, in other words, why do
     many large proteins contain multiple copies of a polypeptide subunit (4 facts required)?                (4)
     ANSWER:………………………………………………………………………………………………………
     ……………………………………………………………………………………………………………………
     …………………………………………………………………………………………………………………

The following questions (7 to 12) are of the multiple choice type (choose the most appropriate answer):

7.   All of the following are considered “weak” interactions in proteins, except:
          a. hydrogen bonds.
          b. hydrophobic interactions.
          c. ionic bonds.
          d. peptide bonds.
          e. van der Waals forces.                                                                          (1)




                                                                                                                    5
8.   In an aqueous solution, protein conformation is determined by two major factors. One is the
     formation of the maximum number of hydrogen bonds. The other is the:
          a. formation of the maximum number of hydrophilic interactions.
          b. maximization of ionic interactions.
          c. minimization of entropy by the formation of a water solvent shell around the protein.
          d. placement of hydrophobic amino acid residues within the interior of the protein.
          e. placement of polar amino acid residues around the exterior of the protein.                  (1)

9.   Which of the following best represents the backbone arrangement of two peptide bonds?
        a. C—N—C—C—C—N—C—C
        b. C—N—C—C—N—C
        c. C—N—C—C—C—N
        d. C—C—N—C—C—N
        e. C—C—C—N—C—C—C                                                                             (1)

10. In an -helix, the R groups on the amino acid residues:
         a. alternate between the outside and the inside of the helix.
         b. are found on the outside of the helix spiral.
         c. cause only right-handed helices to form.
         d. generate the hydrogen bonds that form the helix.
         e. stack within the interior of the helix.                                                      (1)

11. The -keratin chains indicated by the diagram below have undergone one chemical step. To alter the shape of
    the -keratin chains - as in hair waving - what subsequent steps are required?




         a.   Chemical oxidation and then shape remodeling
         b.   Chemical reduction and then chemical oxidation
         c.   Chemical reduction and then shape remodeling
         d.   Shape remodeling and then chemical oxidation
         e.   Shape remodeling and then chemical reduction                                               (1)

12. Which of the following statements is false?
       a. Collagen is a protein in which the polypeptides are mainly in the -helix conformation.
       b. Disulfide linkages are important for keratin structure.
       c. Gly residues are particularly abundant in collagen.
       d. Silk fibroin is a protein in which the polypeptide is almost entirely in the -conformation.
       e. -keratin is a protein in which the polypeptides are mainly in the -helix conformation.       (1)




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