WRITE YOUR NAME AND I.D. NUMBER LEGIBLY ON EVERY PAGE – PAGES WILL BE SEPARATED FOR GRADING! CHECK TO BE SURE YOU HAVE 5 PAGES, NAME (print): INCLUDING COVER PAGE. 5-digit course ID # I swear/affirm that I have neither given nor received any assistance with this exam. Signature: Date: BIOCHEMISTRY 460 SECOND HOUR EXAMINATION FORM A -- ANSWERS March 6, 2006 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 J•mol–1•Kelvin–1 = 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) Henderson-Hasselbalch Equation: ! pH = pKa+ log[base][acid] 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 12.5Biochemistry 460, Exam #2 Form A NAME March 6, 2006 ANSWERS 5-digit course ID# page 2 1. (6 pts, 3 pts each) A. The enzymes that catalyze phosphorylation of specific functional groups on their target proteins by transferring a phosphate group from ATP to a Ser, Thr, or Tyr residue are called protein _kinases__________ . B. The enzymes that catalyze hydrolytic removal of phosphate groups from phosphorylated proteins are called protein __phosphatases________ . 2. (12 pts) On the plot below is the substrate binding curve for an enzyme whose structure is a homotetramer. On that same set of axes, sketch substrate binding curves for the following, and label your curves as E+A, E+B, or C: A. (3 pts) same enzyme (Enz), but in the presence of a heterotropic allosteric activator A (E + A). B. (3 pts) same enzyme (Enz), but in the presence of a heterotropic allosteric inhibitor B (E + B). (Don’t forget parts C and D, below right.) C. (3 pts) a mutant enzyme (C) in which the predominant structure in solution is a dissociated form of the enzyme, individual subunits (monomers) rather than tetramers, with a much higher affinity for substrate than the normal enzyme. D. (3 pts) Indicate on the plot how you would determine the KM value for the monomeric mutant (your curve C). 3. (4 pts) Circle ALL correct statements about P-type ATPases: A. They are membrane proteins whose structures are large β barrels with an aqueous channel through the middle through which a variety of solutes can cross the membrane in both directions. *B. They catalyze an active transport process in which a solute is transported across a membrane against its concentration gradient. C. Their normal mechanism of action involves ATP synthesis driven by ion transport. *D. Their mechanism of action involves a conformational change coupled to phosphorylation and dephosphorylation of an Asp residue. E. None of the above statements is/are correct. 4. (4 pts) Briefly explain why it is important that pancreatic acinar cells (which biosynthesize and secrete zymogens into the digestive system) contain pancreatic trypsin inhibitor (PTI), i.e., what is the biological function of PTI? PTI very effectively inhibits any trypsin that's prematurely activated INSIDE the pancreatic cells, preventing it from "autodigesting" the pancreas from the inside. /26 p. 2 (26 points) p. 3 (30 points) p. 4 (24 points) p. 5 (20 points) TOTAL: (100 points)Biochemistry 460, Exam #2 Form A NAME March 6, 2006 ANSWERS 5-digit course ID# page 3 5. (9 pts) Some membrane proteins, e.g. the GLUT 1 transporter, have multiple membrane spanning amphipathic α helical segments in their structures. A. (4 pts) Which one of the helices shown below in helical wheels representations is an amphipathic α helix? Helix #3 1) 2) 3) 4) B. (5 pts) Briefly explain how an amphipathic helix within the GLUT1 structure would be oriented in the membrane relative to the lipid bilayer and the protein’s binding site for glucose. The hydrophobic side of the helix would be “facing” the hydrophobic core of the lipid bilayer on the outside of the protein and the hydrophilic side would be in contact with the channel/binding site for glucose. 6. (13 pts) The activity of chymotrypsin changes as the pH changes in the range of pH 5-9, as shown on the graph below. From your understanding of the chemical mechanism of chymotrypsin, explain the pH effect on the enzyme’s activity, by answering parts A, B and C below. Specifically, A. (4 pts) What enzyme functional group of what amino acid residue is most likely responsible for the effect of pH? the imidazole of the active site His residue B. (4 pts) What is the apparent pKa of that functional group? Indicate on the graph how you deduced the pKa. pKa is pH at which [base] = [acid], so protein would be 50% active – the pH is 6.8. C. (5 pts) Briefly explain in terms of the chymotrypsin mechanism why the activity increases as the pH increases in this pH range. In the first catalytic step of the chymotrypsin mechanism, the active site His residue has to act as a general base, accepting a proton from the active site Ser, so the His has to start out in its unprotonated form (active form is conjugate base]. 7. (8 pts) Consider the simple reaction: kF(uncat) with no catalyst is 10–5 s–1; kR(uncat) with no catalyst is 10–2 s–1. kF(cat) with enzyme catalyst is 107 s–1. A. (4 pts) What is Keq for the
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