Exam 2, BICH 410, Section 503 (MWF 3-3:50), Monday, October 26, 1998

Write your name on each page. Write concise answers to demonstrate effectively your mastery of the subject material. Show your work in order to receive partial credit where applicable.
gas constant R 8.315 J/mol-K; Faraday constant F 96.5 kJ/mole-volt

1. (25 pts) Provide short answers or fill in the blanks with the best answer.
a) What is meant by the steady-state assumption in the derivation of the Michaelis-Menten equation?
Answer: rate of formation of ES complex = rate of decay of ES complex

b) Give two examples of ionophores __________________, __________________
Answer: valinomycin, gramicidin A

c) How is CO2 (or bicarbonate) carried on the hemoglobin protein? Show a structure.
Answer: binds to amino terminus as a carbamate (structure shown in book on pg. 231)

d) What does it mean that antibodies are bivalent?
Answer: contains two antigen binding sites

e) What is the name of a three-carbon aldose?
Answer: glyceraldehyde

f) What two amino acid residues are the sites of attachment for O-linked glycoproteins?
Answer: serine, threonine

g) What type of membrane protein could be extracted from membranes by changing the pH or increasing the salt concentration (Circle correct answer): peripheral or integral
Answer: peripheral

h) What transporter protein is inhibited by oubain?
Answer: sodium-potassium pump

i) What important component involved in neurotransmission is produced by the Schwann cells?
Answer: myelin (sheath)

j) What are possible units for a second-order rate constant?
Answer: M-1sec-1 or concentration-1time-1

k) Name three examples of serine proteases.
Answer: chymotrypsin, trypsin, subtilisin, elastase

2. (6 pts) Below is the sequence of a portion of a membrane protein. Give three reasons why this sequence is unlikely to constitute an entire transmembrane alpha helix. How would you change the sequence such that it could be a transmembrane alpha helix?
val-phe-leu-asp-pro-ala-ala-ile-leu-val-met

Answer: 1) too short; needs to be 20 amino acids long, so add 9 more hydrophobic amino acids
2) proline is helix-breaker; replace pro with a hydrophobic amino acid (like val)
3) asp is hydrophilic; replace asp with a hydrophobic amino acid (like val)

3. (10 pts) Draw the structure of alpha-D-glucopyranosyl(1 -> 6)beta-D-glucopyranose using a Haworth projection.

Answer: see key

4. (9 pts) Draw the catalytic triad and substrate of chymotrypsin at the stage in the mechanism where there is an oxyanion transition-like state in the deacylation (second) phase.

Answer: see Fig. 11.13, stage 5

5. (15 pts) 20 micrograms of an enzyme with molecular weight 50,000 are used in 1 ml (for each reaction) to generate the Lineweaver-Burk (double reciprocal) plot shown here. Calculate the ratio kcat/KM for this enzyme in units of sec-1M-1.

Answer: Strategy of the solution
Calculate KM from plot. Intersection of extrapolated line with X-axis = -1/KM. KM = 33 micromolar
Calculate Vmax from plot (intersection of extrapolated line with Y-axis = 1/Vmax. Vmax = 154 micromolar/sec.
Since Vmax = kcat[ET], then kcat = Vmax / [ET].
[ET] = (20 ug) / [(50,000 ug/umole)(1 ml)(1 liter / 1000 ml)] = 0.4 micromolar
kcat = 154 micromolar/sec / 0.4 micromolar = 385 sec-1
Then, kcat / KM = 385 sec-1 / (33 micromolar)(1 M / 106 micromolar) = 1.2 X 107 sec-1M-1

6. (9 pts) Sketch two oxygen binding curves for adult hemoglobin on the same plot, one in the absence and one in the presence of BPG. Label the axes of your plot. Also, indicate the P50 values for each binding curve.

Answer: see key; two sigmoidal curves on theta vs. pO2 plot; +BPG curve is shifted to right; P50 values are pO2 where theta is 0.5

7. (12 pts) Draw the structure of phosphatidylserine (a glycerophospholipid) where one fatty acid is 14:0 and the other fatty acid is 18:2cdelta9,12.

Answer: see answer key (also, part of structure is on Fig. 10.7 in book)

8. (14 pts) A) The concentration of Na+ inside a vertebrate cell is about 12 mM and the cell is bathed in blood plasma containing about 145 mM Na+. For a typical cell with a transmembrane potential of -70 mV (inside negative relative to outside), what is the free energy change for transporting 1 mole of Na+ out of the cell at 37 C?
B) What transporter catalyzes this process? Describe the ions transported, the stoichiometry of ion transport, the direction of ion transport, and whether this process requires the hydrolysis of ATP.

Answer: part A) deltaG = RT ln Cout / Cin + ZF(membrane potential)
plug in numbers from problem, making sure both sides of equation are in kJ/mole; the "concentration term" should be positive because transporting against a concentration gradient; the "membrane potential term" should be positive because transporting a positive ion to a more positive side of the membrane
deltaG = 6.43 kJ/mole + 6.76 kJ/mole = +13.2 kJ/mole
part B) sodium-potassium pump
3 sodium ions transported outside the cell and 2 potassium ions transported inside the cell for every ATP hydrolyzed