Biochemistry (Chem 4400)

1. (15 points) Catalase protects the body from oxidation by catalyzing the breakdown of H₂O₂a potentially potent oxidant, to form carbon dioxide and water.

a. If a 1 mL solution of catalase destroys H₂O₂ at a rate of 3.24 moles/min under saturation conditions at 37^oC, and the k_cat for catalase is 2.7 x 10⁹ min^-1, how much enzyme is present (in moles/L)? b. The K_M value for catalase is 1.1M, what is concentration of substrate H₂O₂ where the velocity is 1/4 of V_max?
c. What is the activation energy for the reaction catalyzed by catalase?
d. If an enzyme increases the rate of a reaction by 100, how much did it lower the activation energy of the uncatalyzed reaction?

2. (15 points) Arylhydrocarbon hydroxylase is an important xenobiotic metabolizing enzyme. It catalyzes the hydroxylation of benzopyrene (BP) to form 3-hydroxybenzo(a)pyrene (3-OH BP). This enzyme can be inhibited by Procardia (nifedipine), a drug used to treat angina. The data results from adding nifedipine to an invitro assay for aryl hydroxylase activity. The data is given in both tabular and in graphical form. 1. Please label the graph with the following:

a. Title
b. An arrow to the line for the data with the inhibitor.

a. V_max (no inhibitor)
b. V_max(inhibitor)
c. K_M (no inibitor)
d. K_M (with inhibitor)

3. (15 points) An oligomeric protein has a molecular weight of 72,400 daltons. It is a manganese protein, with 0.076% manganese (the atomic weight of Mn is 54.94) When 868 mg of the protein is reacted with 1-fluoro-2,4-dinitrobenzene and the peptide bonds completely hydrolyzed with 6N HCl at 100 C overnight, 2.86 mg of the 2,4 dinitro derivative of threonine was produced and 3.3 mg of the 2,4 dinitro derivative of methionine was also produced.

a. How many manganese atoms are present in each oligomeric protein molecule?
b. How many subunits are present in the oligomeric protein?

4. (10 points) Keratin is the major protein in wool from sheep. On average, sheep wool grows 60 cm per year. The fundamental structure of wool is keratin, a rope like protein that consists primarily of alpha helices. The alpha helix contains 3.6 amino acids per turn with a rise of 0.56 nm per turn. (In other words each 0.56 nm of hair contains 3.6 amino acids.) The rate determining step for hair growth is the formation of peptide bonds in the keratin chains. Calculate the rate of peptide bond formation in the keratin chains that would correspond to the observed yearly hair growth.

5. (10 points) Many enzyme reaction mechanisms involve the transfer of protons from amino acids in the protein to the substrate.

a. Given the following pH values for optimum enzymatic activity, predict what amino acids could be involved in the enzyme mechanism. For this part, use the standard pKa values that you have memorized.

b. Electronic effects within a protein often change the pKa values for the ionizable groups on amino acids. Explain (using structures) how glutamate could have pKa=2 in a protein.

6. (10 points) Many enzymes must bind two substrates to catalyze the formation of a product. Often the binding of one substrate will affect the binding of the other substrate.

a. Using the expression below for Enzyme (E) binding substrates (A) or (B) or both, and the binding constants K_A = 4 x 10^-5, K^{B = 3 x 10-4, K_BA = 2 x 10-6, determine the value for K_AB.
b. Does the binding of A enhance the binding of B?}

7. (15 points)Benzo(a)pyrenes are present as the incomplete combustion products of fossil fuel buring. They are converted to reactive epoxide molecules by the enzyme, p450. These reactive molecules react with guanine nucleotides to cause DNA damage. An enzyme is needed to repair this damage. Consider the following data:

1. The enzyme hydrolyzes the 3' to 5' phophodiester bond of DNA that contains a (3') Guanosine that has been modified with a large hydrophobic group (at N7) such as the benzo(a)pyrene metabolites.
2. The enzyme is an oliomeric protein with two subunits and one Manganese atom per enzyme molecule.
3. The optimal pH is 7.
4. Using x-ray crystallography, the following amino acids were found to surround a nonhydrolyzable substrate: R, Y, G, L, D, E, H, S

a. Suggest a possible nonhydrolyzable structure for use in structure determinations.
b. Draw the amino acids around the substrate to show how the protein stablilizes the transition state of the substrate.
c. Suggest a possible mechanism for this enzyme.

8. (10 points)Choose one of the following questions, and answer it using complete sentences. Be sure to that your answer is well organized and clearly written.

I. Explain how some genetic mutations affect all three types of hemoglobin A, A₂ and F, and some mutations effect only one of these types of hemoglobin.

II. Equation 3 below was derived from equation 1 by substituting P50 for the Equilibrium constant, K, into equation 2 below. Rationalize this substitution(why does P50 = K?). Also, the Hill plot for hemoglobin has a slope of 2.8 near the midpoint (Y = 0.5). Near the ends of the graph, at high oxygen pressure or at very low oxygen pressure, the slope approaches 1. Explain this phenomena.

III. Discuss how the structural elements of an enzyme are involved in catalyzing reactions.