WRITE YOUR NAME LEGIBLY ON EVERY PAGE – PAGES WILL BE SEPARATED FOR GRADING! CHECK TO BE SURE YOU HAVE 5 PAGES, NAME (print): INCLUDING COVER PAGE. I swear/affirm that I have neither given nor received any assistance with this exam. Signature: Date: BIOCHEMISTRY 460 SECOND HOUR EXAMINATION FORM B (white) -- ANSWERS March 10, 2008 A NON-PROGRAMMABLE CALCULATOR MAY BE USED ON THIS EXAM. No programmable calculators are permitted, and no sharing of calculators. We have a couple of spare calculators to lend in an emergency. SHOW YOUR WORK FOR ALL CALCULATIONS, AND BE SURE TO STATE UNITS OF ANY NUMERICAL ANSWERS. If the reasoning, calculations, or answer are shown anywhere other than in the space provided, make a note in the space provided and put answer on BACK OF SAME PAGE so the grader for that page will have it. USEFUL CONSTANTS: R (gas constant) = 8.315 x 10–3 kJ•mol–1•Kelvin–1 If temperature = 25 °C, absolute temperature T = 298 K (Assume this temperature unless problem states otherwise.) Potentially useful equations: Michaelis-Menten Equation: Vo= Vmax[S][S] + Km Free energy of transport: ! "Gt= RT lnC2C1+ zF"V where F is the Faraday constant (96.5 kJ/V•mol), z is the charge on the solute, and ΔV is the membrane potential (charge gradient across the membrane) Use these “generic” pKa values only when no precise pKa for a specific group is given. Ionizable group in peptides and proteins Approximate ("generic") pKa in peptides & proteins (from Berg, Tymoczko & Stryer, Biochemistry, 5th ed., 2001) α-carboxyl 3.1 side chain carboxyl 4.1 imidazole 6.0 α-amino 8.0 thiol 8.3 aromatic hydroxyl 10.9 ε-amino 10.8 guanidino 12.5 p. 2 (21 points) p. 3 (30 points) p. 4 (24 points) p. 5 (25 points) Total (100 points)Biochemistry 460, Exam #2 Form B NAME March 10, 2008 ANSWERS page 2 1. (8 pts) Circle T (true) or F (false) to indicate if each of the following statements is true or false. T F Protein kinase A phosphorylates Ser and Thr residues on its target proteins. T F Decreasing the chainlength of the fatty acids in membrane lipids would increase fluidity. T F Increasing the number of double bonds in membrane fatty acid chains would increase fluidity. T F A single transmembrane helix that is amphipathic would be an excellent way for an integral membrane protein to cross the hydrophobic core of the lipid bilayer. 2. (13 pts) The enzyme papain is a protease that uses a mechanism similar to the mechanism used by chymotrypsin, except that there is an active site Cys in papain carrying out the catalytic role played by the active site Ser of chymotrypsin. There is a His residue in papain that plays the same role as the active site His residue in chymotrypsin. A. (4 pts) The structure of 2 residues of a peptide substrate for papain is shown below. Which of the structures below (1-6) would be the structure of the first tetrahedral intermediate in the papain-catalyzed hydrolysis of the peptide bond in that substrate? Answer: (4) B. (9 pts) 1) Name the region of the chymotrypsin active site responsible for tight transition state binding in chymotrypsin’s chemical mechanism. “oxyanion hole” 2) Like chymotrypsin, papain should also have a region responsible for binding/stabilizing the transition state in its peptide hydrolysis mechanism. Use your knowledge of the mechanism of chymotrypsin to name the structural element of the transition state that would be tightly bound by papain. (You don’t need to draw a structure.) negatively charged O atom (O–) (the oxyanion) of the tetrahedral transition state 3) What type of enzyme functional groups might be used to bind the transition state for papain? There’s more than one correct answer; suggest only ONE. To stabilize the oxyanion of the tetrahedral intermediate, papain might use: •hydrogen bonding to two backbone N-H groups (this is what chymotrypsin does), or •a metal ion (e.g., Zn2+ or Mg2+), or •a nearby positively charged group, e.g. Lys (ε-amino) or Arg (guanidino) or α-NH3+. /21Biochemistry 460, Exam #2 Form B NAME March 10, 2008 ANSWERS page 3 3. (3 pts) The adenylate kinase cascade that includes the activation of protein kinase A results in phosphorylation of both the enzyme that makes glycogen and the enzyme that breaks down glycogen. Phosphorylation of one of the enzymes results in its activation, and phosphorylation of the other results in its inactivation. This kind of opposing regulation of opposing biochemical pathways is called __reciprocal____ regulation. 4. (9 pts) The figure below shows the pathway for biosynthesis of the purine nucleotides AMP and GMP, with the enzyme catalyzing each step designated as E1, E2, etc. A. (3 pts) If the concentration of AMP but not GMP were getting too high in the cell, which enzyme would most likely be feed-back inhibited by AMP? __E8___ B. (3 pts) If the concentration of GMP but not AMP were getting too high in the cell, which enzyme would most likely be feed-back inhibited by GMP? __ E6__ C. (3 pts) If the concentrations of BOTH purine nucleotides, AMP and GMP, were getting too high in the cell, which enzyme would most likely be inhibited by binding of both AMP and GMP to slow the overall rate of purine nucleotide biosynthesis? __ E3__ (1st committed step) 5. (18 pts, 2 pts each) Matching: Choose one answer from the choices below (1-12) for each blank in parts A-I. Some answers will not be used. Give only one answer per blank. 1) arachidonate 4) protein kinase A 7) bacteriorhodopsin 2) HIV protease 5) phospholipase A2 8) thrombin 3) calmodulin 6) prostaglandin 9) COX (PGH2 synthase) 11) 10) 12) Put the number of just one choice from above in each blank below. A. 3 protein that binds Ca2+ with EF hand structural motif B. 8 protein that binds Ca2+ with γ-carboxyglutamate residues C. 11 a sphingolipid D. 1 substrate for cyclooxygenase (COX, PGH2 synthase) E. 4 enzyme activated by cyclic AMP F. 9 enzyme inhibited by aspirin and ibuprofen to reduce inflammation G. 12 a glycerophospholipid H. 5 enzyme inhibited by corticosteroids to reduce inflammation I. 10 a steroid /30Biochemistry 460, Exam #2 Form B NAME March 10, 2008 ANSWERS page 4 6. (24 pts)
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