Problem Set on Binding Equilibrium and Enzyme Kinetics 1. Cytochrome f operates as a redox agent in chloroplast photosynthesis.

The standard reduction potential, , of cytochrome f at pH 7.0 is determined by coupling it to an agent of known such as ferricyanide ferrocyanide. In a typical experiment carried out spectrophotometrically, a solution at 25C and pH 7.0 containing a ratio, [Fe(CN)64 ] / [Fe(CN)63 ] equal to 2.0 is found to have a ratio [Cyt f reduced]/[Cyt f oxidized] of 0.10 at equilibrium. a. Calculate the reduction potential for Cyt f. b. On the basis of the standard reduction potential, , for the reduction of O2 to H2O at pH 7 and 25C, is oxidized Cyt f a good enough oxidant to cause the formation of O2 from H2O at 25C and pH 7? The enzyme nucleoside diphosphokinase catalyzes the reaction: Mg2+ GTP + dGDP GDP + dGTP In an experiment with the ezyme isolated from erythrocytes, the following results were obtained: [GTP] (M) 30 50 Velocity (katal kg 1) 20 0.095 0.112 0.141 25 0.102 0.120 0.155 40 0.112 0.136 0.180 100 0.125 0.156 0.218 What can you deduce from these data regarding a likely mechanism for the catalyzed reaction? How could you check your conclusion? [dGDP] (M) 3. 22 200 0.196 0.223 0.284 0.385 enzyme

2.

The binding of a ligand A to a macromolecule was studied by equilibrium dialysis. In each measurement a 1 M solution of the macromolecule was dialyzed against an excess amount of a solution containing A. After equilibrium was reached, the concentration of A on each side of the dialysis membrane was measured and the ff data obtained. Total concentration of A Solvent Side (M) Macromolecule side(M) 5.1 5.7 10.2 12.8 20.1 23.4 52.2 56.2 105.0 109.1 200 204.7 Determine the binding constant and the total number of binding sites.

4.

The following data were obtained for the variation of Vmax with pH for the fumarase reaction at 298 K (Vmax in arbitrary units) pH 5.2 5.5 6.0 6.5 6.75 7.0 7.5 8.0 Vmax 54 105 184 224 236 230 162 86 Deduce the appropriate pK values.

8.5 33

5.

The following data were obtained for the myosin catalyzed hydrolysis of ATP: Temperature (C) k x 106 (sec 1) Calculate Ea. 39.9 4.67 43.8 7.22 47.1 10.0 50.2 13.9

6.

The equilibrium: I2 + I I3 1 , has been studied using a laserinduced temperature jump technique. Relaxation times were measured at various equilibrium concentrations of reactants at 25C: [I 1] (mM) 0.57 1.58 2.39 2.68 3.45 [I2] (mM) 0.36 0.24 0.39 0.16 0.14 71 50 39 38 32 (ns)

Calculate the constants k1 and k1 for the system at 25C.