Part I: [42 pts]: For multiple choice, choose the one BEST answer. Circle its letter. Read each question and all 5 choices carefully because more than one answer might seem correct at first glance. 2 pt each

1. During gel electrophoresis, larger molecules move
a. slower than smaller molecules
b. farther than smaller molecules
c. according to their size, while smaller molecules move according to their net charge
d. in the opposite direction from smaller molecules
e. any of these may be true, depending on the type (or conditions) of electrophoresis

2. A carbonium ion
a. is C⁺
b. is highly reactive
c. is typical of both enzymatic and non-enzymatic biological reactions
d. a and b
e. all of the above

3. Compare to that of myoglobin, hemoglobin affinity for oxygen is
a. higher
b. lower
c. higher in muscle capillaries, lower in lung
d. lower in muscle capillaries, higher in lung
e. can not be measured or compared

4. DG
a. if > 0 the reaction is spontaneous
b. depends on concentration of reactants, not products
c. depends on concentration of products, not reactants
d. depends on free energy of reactants and products, independent of reaction pathway
e. depends on free energy of reactants, transition state, and products

5. k₂ + k₃
a. is the rate of ES formation
b. = k₁
c. is the rate of product formation
d. is the rate of ES loss
e. = K_m

6. Which of the following is NOT true of sickle cell anemia?
a. the heterozygous state may be advantageous in malarial regions
b. it results from a point mutation (a single base pair)
c. it results from a single amino acid substitution
d. it is an example of a frameshift mutation
e. there is variation of expression among heterozygous individuals

7. Which of the following is FALSE?
a. as K_m increases, E-S binding strength or affinity increases
b. low K_m means a small dissociation rate
c. K_m is always > 0M
d. K_m is usually <1M
e. all of the above are TRUE

8. At high [S], V depends mostly on:

9 At low [S], V depends mostly on:

10. If DG = 0 for A + B <==> C + D, what will accumulate?

11. Lysozyme
a. hydrolyzes all of the NAG-NAG bonds in (NAG)₆
b. fills about half of the active site cleft with (NAG)₃
c. cleaves (NAG)₄, (NAG)₅, and (NAG)₆ at about the same rate
d. all of the above
e. none of the above

12. Which of the following is NOT a serine protease?
a. chymotrypsin
b. subtilisin
c. thrombin
d. trypsin
e. lysozyme

13. Chymotrypsin activity characteristics include
a. the catalytic mechanism involves acylation/deacylation
b. a transient covalent bond is formed between the substrate and the enzyme
c. Zn⁺⁺ is critical for substrate binding
d. a and b
e. all of the above

14. Which of the following are true of enzymes?
a. a reaction will reach equilibrium faster with enzyme catalysis than without
b. the tertiary structure of an enzyme is more important for function than its primary structure
c. enzyme activity can affect the Gibbs free energy of a reaction’s transition state
d. a and b
e. all of the above

15. Tri-NAG binds the lysozyme active site via
a. H-bonds
b. Van der Waals forces
c. electrostatic forces
d. a and b
e. all of the above

16. Endopeptidase
a. are used in amino acid sequencing
b. are not ordinarily found in nature
c. cleave a polypeptide sequentially from one end or the other
d. cleave proteins but not polypeptides
e. all of the above

17. pH is an important issue for enzyme activity because
a. pH affects the ionization state of potential H⁺ donors and acceptors
b. pH affects protein tertiary structure
c. pH is only important when electrostatic bonds are used for substrate binding
d. a and b
e. all of the above

18. Which of the following is NOT likely to affect V_max?
a. temperature
b. [S]
c. pH
d. phosphorylation of the enzyme
e. enzyme degradation

19. Which of the following is NOT likely to affect K_m?
a. temperature
b. [S]
c. pH
d. phosphorylation of the enzyme
e. substrate analogs

20. It is inferred that the glutamic acid 35 residue, rather than aspartic acid 52 donates its H⁺ during NAG hydrolysis by lysozyme at the enzyme’s pH optimum because
a. only glu is ionized at pH5
b. only asp is ionized at pH 5
c. glu is ionized, while asp is not ionized, due to local effects of other, surrounding amino acid residues
d. asp is ionized, while glu is not ionized, due to local effects of other, surrounding amino acid residues
e. asp is facing the wrong way

21. SDS polyacrylamide gel electrophoresis
a. denatures proteins
b. coats proteins so that their charge is approximately proportional to their molecular mass
c. separates proteins according to size
d. a and b
e. all of the above

Part II. [18 pts] Short answer. Choose 3. 6 points each

1. One of the first steps in protein sequencing is determining the number of different polypeptide chain subunits comprising the protein. Briefly describe the methods used for this analysis. Be sure to mention AT LEAST two different approaches used.

2. Identify three techniques for N-labeling. Briefly explain the advantages and disadvantages of each.

3. Why is it necessary to fragment a purified polypeptide before amino acid sequencing?

4. Why is it necessary to fragment a polypeptide in more than one fashion, and sequence each set of fragments. Illustrate this approach using a theoretical example.

5. Briefly describe two different techniques for isolating (purifying) and characterizing a polypeptide. What characteristics can, and which can not be inferred from the two techniques you chose?

Part III. [12 pts] Short answer. Choose 2. 6 points each

1. Sketch a kinetic plot (V x [S] ) of an enzyme + a competitive inhibitor. Sketch the corresponding Lineweaver-Burk plot. Explain the effect of the inhibitor - why does it affect the kinetics in this way?

2. Sketch a kinetic plot (V x [S] ) of an enzyme + a non-competitive inhibitor. Sketch the corresponding Lineweaver-Burk plot. Explain the effect of the inhibitor - why does it affect the kinetics in this way?

3. Under what circumstances will an enzyme's kinetic V x [S] plot be hyperbolic, and when sigmoidal? Explain.

Part IV. [28 pts] Shorter answer. 4 points each

1. Enzymes accelerate reactions by
a.
b.

2. How does saturation demonstrate the existence of ES? (What alternative is disproved by saturation?)

3. K_m= . Explain the significance or meaning of this.

4. K_m= . (different from #3) Explain the significance or meaning of this.

5. Under what circumstances, if any, can a substrate and an inhibitor bind a enzyme simultaneously. What is the significance of this?

6,7. Compare the models of allosteric cooperativity illustrated by ATCase (concerted model) and hemoglobin (sequential model). [Bonus .. 1 pt: Is it important to this discussion that the number of allosteric components, binding sites, per protein unit is different in the two examples? Explain.]

EXTRA CREDIT: Write you own question and answer it. HINT: this is you opportunity to show what you know. Do not ask yourself a question if you aren't sure of the answer. Ask the question you were sure would be on the exam, so you studied the material, and then somehow it was left off! Your score will depend on the quality of the question as well as the answer - up to a possible maximum of 5 pts.