BCH 4053 PRE-TEST 2 GROUP NAME _____________________ June 19, 1996 This test is take-home and open book, and it is intended that all members of the group contribute to completing it. Only one copy is to be submitted by the group, and all members who participated should sign their names below. Test is due by 1:30 pm on Monday, June 24. __________________________ __________________________ __________________________ __________________________ __________________________ __________________________ Points (23) 1. A biochemist studying the properties of a newly isolated metabolic enzyme obtains the following rate data during kinetic experiments in the absence and presence of two different inhibitors, A and B, one a substrate analogue. [S] v v v (M x 104) ęmol/min ęmol/min ęmol/min 5.0 1.25 0.74 0.48 2.5 0.87 0.45 0.33 1.7 0.67 0.32 0.25 1.2 0.54 0.25 0.20 1.0 0.45 0.21 0.17 Inhibitor A concentration is 5 x 10-4 M; that of B is 3.2 x 10-6 M. a. Determine Vmax and KM for this enzyme using the Lineweaver-Burk reciprocal plot. Plot the inhibitor data on the same graph. (Note: Pick your axes and scales carefully so that the lines may be extrapolated to the negative x intercept. It would be a good idea to draw the graph on scratch graph paper first, then do a clean finished copy.) b. Classify the inhibitors as competitive, not-competitive, non- competitive, or uncompetitive. Which one is likely to be the substrate analogue? c. Plot the data for the uninhibited reaction on a separate graph using the Eadie-Hofstee plot (v plotted versus v/S). Determine Vmax and Km for the uninhibited reaction from this graph, and compare with part a. (Show your calculations on a blank sheet of paper, and plot your graphs on the graph paper, and attach them to the exam. Put your group name on the sheet and the graph paper.) (12) 2. Following are three models for reversible inhibition of a simple one-substrate enzyme reaction, three rate laws which are derived from the models, expressed in reciprocal form, and four terms describing types of reversible inhibition. For the graphs which follow, indicate in the blank below the graph the model (a, b, or c), the rate law (d, e, or f), and the term(s) (g, h, i, or j) which apply to that graph. (a) E + S ž ES E + P (b) E + S ž ES E + P E + I ž EI ES + I ž ESI (c) E + S ž ES E + P where (for a, b, and c): E + I ž EI KI = [E][I]/[EI] ES + I ž ESI K'I = [ES][I]/[ESI] (d) 1/v = (1/Vm)(1 + I/K'I) + (Km/Vm)(1/S) (e) 1/v = (1/Vm)(1 + I/K'I) + (Km/Vm)(1/S)(1 + I/KI) (f) 1/v = (1/Vm) + (Km/Vm)(1/S)(1 + I/KI) (g) competitive (h) not-competitive (i) non-competitive (j) un-competitive ³ ³ ³ lines ³ 1/v ³ intersect on 1/v ³ parallel ³ x axis ³ lines ³ ³ ³ ³ ÄÄÄÄÄĮÄÄÄÄÄÄÄÄÄÄÄÄÄÄ ÄÄÄÄÄĮÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄ 1/S 1/S _________________ ________________ ³ ³ ³ lines ³ lines 1/v ³ intersect on 1/v ³ intersect off ³ y axis ³ axes ³ ³ ³ ³ ÄÄÄÄÄÄĮÄÄÄÄÄÄÄÄÄÄÄÄÄÄ ÄÄÄÄÄĮÄÄÄÄÄÄÄÄÄÄÄÄÄÄ 1/S 1/S ________________ _________________ (12) 3. Subtilisin (MW = 27,600) is a bacterial protease that can catalyze hydrolysis of certain amino acid esters and amides. For the synthetic substrate N-acetyl-L-tyrosine ethyl ester (Ac-Tyr-OEt), subtilisin exhibits Km and kcat (or k2 as used in lecture) values of 0.15 M and 550 s-1, respectively. (a) What is the Vmax for the hydrolysis of Ac-Tyr-OEt when the subtilisin concentration is 0.4 mg ml-1? (b) Indole is a competitive inhibitor of subtilisin with a Ki of 0.05 M. What is the Vmax for Ac-Tyr-OEt hydrolysis by 0.40 mg ml-1 subtilisin in the presence of 6.25 mM indole? (c) What is the velocity (v) when 0.40 mg ml-1 subtilisin is incubated with 0.25 M Ac-Tyr-OEt? (9) 4. Classify the following fatty acids as ž-9, ž-7, ž-6, or ž-3. 9-C18:1 ________ 5,8,11,14-C20:4 _________ 9-C16:1 _______ 9,12-C18:2 _______ 9,12,15-C18:3 _________ 8,11,14-C20:3 _______ (8) 5. Circle the following lipids which are negatively charged at pH 6. phosphatidyl choline phosphatidyl serine phosphatidyl glycerol cholesterol sphingomyelin palmitic acid phosphatidic acid phosphatidyl inositol (12) 6. The mechanism of chymotrypsin illustrates several of the factors that are believed to contribute to the rate acceleration obtained by enzymes. Describe each of the following aspects of the chymotrypsin mechanism. (a) A reaction model that shows ping-pong kinetics. (i.e., identify the components of the ping-pong mechanism, figure 8-13b, page 219). (b) Transition state stabilization by bonds formed between the enzyme and the transition state that are not found in the binding of substrate or product. (c) Acid-base catalysis mediated through a "catalytic triad". Describe how the triad assists in the formation of the covalently bound intermediate. (d) Substrate specificity provided by the nature of the substrate binding site. (Explain how chymotrypsin differs from trypsin in the binding site.) (9) 7. Draw the structures of the products from the following reactions: (a) mild acid hydrolysis of choline plasmalogen. (b) mild base hydrolysis of phosphatidyl serine (c) phospholipase A hydrolysis of phosphatidyl glycerol (6) 8. Describe three types of lipid anchors that attach some proteins to membranes. (9) 9. Distinguish between integral and peripheral membrane proteins in terms of (a) types of solutions used to extract them from membranes. (b) forces by which they are attached to membranes. (c) membrane location in the fluid mosaic model.