Lecture 13: Data Analysis for the V versus [S] Experiment and Interpretation of the Michaelis-Menten Parameters

Transcription

1 Biological Chemistry Laboratory Biology 3515/Chemistry 3515 Spring 2018 Lecture 13: Data Analysis for the V versus [S] Experiment and Interpretation of the Michaelis-Menten Parameters 20 February 2018 c David P. Goldenberg University of Utah goldenberg@biology.utah.edu

2 Analysis of Data from the V versus [S] Experiment Velocity Substrate Concentration We want to fit the experimental data to the Michaelis-Menten Equation: V = [S]V max [S] + K m From the fit, we obtain estimates of K m and V max.

3 Clicker Question #1 Estimate V max from the graph: nm/s 2 40 nm/s nm/s 4 80 nm/s close enough for credit

4 Clicker Question #2 Estimate K m from the graph: µm 2 2 µm µm 4 10 µm

5 A Classic Method for Analyzing Enzyme Kinetics Data Rearrangement of the Michaelis-Menten Equation: V = [S]V max [S] + K m 1 V = [S] + K m [S]V max = [S] [S]V max + 1 V = 1 [S] K m + 1 V max V max K m [S]V max A plot of 1/V versus 1/[S] should generate a straight line with a slope of K m /V max and an intercept of 1/V max on the 1/V axis.

6 The Lineweaver-Burk Plot 0 0 If the data are perfect, this plot gives good estimates of K m and V max. But, experimental error in V can lead to strange effects!

7 Experimental Error and Uncertainty Error bars for rate measurements are of approximately constant size (e.g., ±0.005 A/min), rather than a constant percentage of the measurement. 0.1 Rate (A/min) For 0.1 A/min, ±0.005 A/min = ±5%. For 0.01 A/min, ±0.005 A/min = ±50%. Enzyme Concentration Least-squares fitting works well if the absolute uncertainties of all data points are approximately equal.

8 What Happens When We Take Reciprocals? V = 0.1 ± = 9.52, = 10.5, 1 V = 10 ± 0.5 V = 0.01 ± = 66.7, = 200, 1 V = 100 ± 50 The values of 1/V derived from small velocities can have huge absolute errors.

9 The Effects on a Lineweaver-Burk Plot Errors in the least precise measurements (low V ) can cause large changes in the line fit to the Lineweaver-Burk plot.

10 Clicker Question #3 Which parameter is likely to be more sensitive to errors in a Lineweaver-Burk plot? 1 K m 2 V max Provided that values of V approach V max.

11 Two Ways to Deal with This Problem Use Lineweaver-Burk, but weight data according to uncertainties in 1/V. Fit velocity data directly to the Michaelis-Menten equation using non-linear least-squares method. Velocity Equal errors in V are weighted equally. Substrate Concentration

12 Interpreting K m E + S E. S E + P Velocity (M/min) K m = k 1 + k cat k 1 V = V max 2 when [S] = K m [S] (M) When [S] = K m, half of total enzyme has substrate bound. The larger K m is, the more substrate is required to reach V max /2, or any specified fraction of V max.

13 Clicker Question #4: Data for three substrates with the same enzyme Which substrate binds most tightly to the enzyme? No wrong answers, for now.

14 A Closer Look at Binding and K m : K m versus K d E + S E. S E + P K m is defined in terms of the rate constants: K m = k 1 + k cat k 1 K d is the equilibrium constant for dissociation. K d = [E][S] [E S] = k 1 k 1 A large K d indicates weak binding.

15 K m versus K d K m = k 1 + k cat k 1 K d = [E][S] [E S] = k 1 k 1 If k cat k 1, i.e., the E S complex is more likely to dissociate than undergo catalysis: K m k 1 k 1 = K d In general, K m K d Strength of equilibrium binding may be greater than indicated by K m.

16 Energy Profile for an Enzyme-Catalyzed Reaction transition state Free-energy change for binding: G bind = RT ln K d R = Gas constant T = Temperature E + S E. S Free-energy change from E S complex to transition state: ( ) G kb T = RT ln k cat h E + P k b = Boltzmann constant h = Planck constant

17 The Significance of k cat /K m E + S E.S E + P Free-energy difference between E + S and the transition state: ( ) G kb T = RT ln +RT ln total h }{{} Constant If k 1 k cat, K d K m : G total = C RT ln ( kcat K m ) ( Kd k cat ) The ratio k cat /K m reflects the free energy difference between E + S and the transition state. (Assuming K d K m ) k cat /K m is commonly interpreted as a measure of enzymatic efficiency. Catalytic efficiency is favored by a large value of k cat and a small value of K m.

18 k cat /K m Is Also the Apparent Second-Order Rate Constant at Low Substrate Concentrations If [S] K m : Velocity (M/min) V = [S][E] T k cat K m + [S] V k cat K m [S][E] T Units of k cat /K m [S] (M) s 1 M = sec 1 M 1

19 Is a Low K m Always Good? Suppose that we could design a mutant enzyme that forms a more stable complex with substrate. wild type E + S E. S mutant E + P This will lower K m and k cat, but leave k cat /K m the same.

20 Clicker Question #5 At low substrate concentration ([S] K m ), will the velocity for the mutant enzyme be greater or less than that of the original enzyme? 1 Greater than the original enzyme wild type 2 Less than the original enzyme 3 The same as the original enzyme! mutant E + S E. S V = [S][E] T k cat K m + [S] E + P V k cat K m [S][E] T

21 What About High Substrate Concentrations? Low Substrate Concentration transition state High Substrate Concentration transition state E + S E. S E. S E + S E + P E + P When [S] K m, the enzyme-substrate complex is favored with respect to the free enzyme.