Velocity, activity units/mg protein 51 3 2 0 5 15 20 Aspartate concentration, mM - 10 Control With With CTP With ATP CTP 25 Figure 2: Kinetics of ATCase in the presence of ATP and CTP (based on Gerhart and Pardee, 1962). The kinetics of the ATCase reaction were examined using increasing concentrations of aspartate, in the presence and absence of CTP and ATP as shown in Figure 2. a. What information can you obtain by looking at the shapes of the curves in this figure? b. What kinetic parameter(s) change in the presence of CTP? What parameter(s) do not change? What is the significance of these observations? c. Answer question 2b for ATP.

Velocity, activity units/mg protein 51 3 2 0 5 15 20 Aspartate concentration, mM - 10 Control With With CTP With ATP CTP 25 Figure 2: Kinetics of ATCase in the presence of ATP and CTP (based on Gerhart and Pardee, 1962). The kinetics of the ATCase reaction were examined using increasing concentrations of aspartate, in the presence and absence of CTP and ATP as shown in Figure 2. a. What information can you obtain by looking at the shapes of the curves in this figure? b. What kinetic parameter(s) change in the presence of CTP? What parameter(s) do not change? What is the significance of these observations? c. Answer question 2b for ATP.

Biochemistry

6th Edition

ISBN:9781305577206

Author:Reginald H. Garrett, Charles M. Grisham

Publisher:Reginald H. Garrett, Charles M. Grisham

Chapter19: The Tricarboxylic Acid Cycle

Section: Chapter Questions

Problem 19P

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Initial rate data for an enzyme that obeys Michaelis–Menten kinetics areshown in the following table. When the enzyme concentration is 3 nmolml-1, a Lineweaver–Burk plot of this data gives a line with a y-intercept of0.00426 (μmol-1 ml s). (a) Calculate kcat for the reaction.(b) Calculate KM for the enzyme.(c) When the reactions in part (b) are repeated in the presence of 12 μM ofan uncompetitive inhibitor, the y-intercept of the Lineweaver–Burk plotis 0.352 (μmol-1 ml s). Calculate K′I for this inhibitor.
Standard redox potentials Eo' for some common compounds in biochemistry: Eo' (Volt) + 0,81 +0,42 + 0,25 12 O₂ + 2 H+ + 2 e NO3 + 2 H+ + 2 e 2 cyt c (ox) + 2 e 2 cyt b (ox) + 2 e Pyruvate + 2 H+ + 2 e* NAD + 2 H+ + 2 e Acétoacétate + 2 H+ + 2 e (2) acétoacétate + NADH + H+ H₂O NO₂ + H₂O 2 cyt c (red) 2 cyt b (red) Lactate (3) 2 cyt c (ox) + 2 cyt b (red) NADH + H+ B-hydroxybutyrate + 0,08 - 0,19 With the help of the above table, determine the spontaneous direction of the following reactions in the standard conditions. Justify your answers by calculating the Gibbs (free) energy of reaction in each case. (1) pyruvate + ß-hydroxybutyrate -0,32 - 0,35 lactate + acétoacétate B-hydroxybutyrate + NAD+ 2 cyt c (red) + 2 cyt b (ox)
Begining with 1 M concentrations of each reactant and product at pH=7 and 25.0 degrees C, calculate the K'eq (to one decimal point) of the reaction Pyruvate + NADH+H+ <=> Lactate + NAD+.Note the temperature of this reaction will not affect the standard reducton potential delta E'o in the table 13-7b. please provide a comprehensive explanation with each step taken.
6-25 substrate-band enzyme concentrations. The the turnover number is equal to umax- b) V=Umax •57(Km+S) anstont For an enzyme that displays Michaelis-Menten kinetics, what is the reaction velocity, V (as a percentage of Vmax), observed at the following values? a) [S] = KM C) d) e) [S] = 0.5KM [S] = = 0.1KM [S] = 2KM [S] = 10KM w reactores -maximumrate of reaction boteles conc. Would you expect the structure of a competitive inhibitor of a given enzyme to be similar to that of its substrate?
CALCULATIONS OF THE SOLUTIONS FOR EXPERIMENT Substrates Stock concentations Succinate + glutamatate (G/S) 150 mM Glutamtate + 150 mM Succinate Malate + pyruvate (M/P) 30 mM Malate + 30 mM Pyruvate Inhibitors Substrates of ETC ATP stock (50 µM): Make pre-dilutions with 20, 15, 10, 5, 2,5 and 1,25 μM ATP (1000 μL of each) Rotenone Oligomycin CCCP KCN Antimycin A Calculate the needed volumen of the standards! Inhibitors of ETC Stock concentations 400 µg/ml in DMSO 1250 µg/ml in EtOH 1000 µg/ml in DMSO 400 mM in Tris-buffer 1 mg/ml in DMSO Calculate the needed volumen! Final concentrations (in 250 μL) 8 μg/ml 25 µg/ml 20 µg/ml 8 mM 20 µg/ml
Begining with 1 M concentrations of each reactant and product at pH=7 and 25.0 degrees C, calculate the K'eq of the reaction Pyruvate + NADH Lactate + NADH+H+. Note the temperature of this reaction will not affect the standard reducton potential delta E° in the table 13-7b.
Many years later, in 1989, Wild, et al. revisited the idea of allosteric control of ATCase by CTP. They noted that CTP did indeed inhibit ATCase, but that the inhibition was always incomplete, even at high concentrations of CTP. They hypothesized that perhaps CTP did not act alone, but in combination with some other nucleotide. They tested the activity of ATCase in the presence of several nucleotide combinations. The results are shown in Table 3. a. What combination gives the most effective inhibition? b. What is the physiological significance of this combination? c. Revise Figure 1 to include this new information. d. Redraw Figure 2 to include this new information. How does the combination compare with the values for the nucleotides alone? Table 3: Relative specific activities for combinations of nucleotide effectors. A value greater than one indicates stimulation, a value less than one indicates inhibition. Nucleotide Effector ATP CTP GTP UTP ATP/CTP ATP/GTP ATP/UTP CTP/GTP CTP/UTP…
Explain the difference between ΔG°’ and ΔG and calculate the free energy (delta G) of hydrolysis of ATP in a rat liver cell in which the ATP, ADP, and Pi concentrations are 3.4, 1.3, and 4.8 mM.
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