Question

ISU Question 3: Use the data below to construct a Michaelis-Menton curve of velocity vs. [S]. This is quite easy to do in Excel. Vo 1/[S1 1/V0 UM (UM/s) M (s/uM) 340 10 2.94E-03 0.2 530 740 0.8 910 1.6 1040 0.4 a) Estimate Vmax from your curve. b) Describe any difficulty you have in completing part (a). Is the enzyme saturated at the highest (SD? c) Using your Vmax estimate, calculate 14 Vmax, and using your curve, estimate Km. Do all of the above from the M-M plot, NOT the L-B plot that you are doing next! c) Now, using your spreadsheet, construct a Lineweaver-Burk (double-reciprocal) plot. Add a linear trendline and equation of the line. d) What is the equation of the line? What does the intercept tell us? What is Vmax? What does the slope tell us? h) What is Km? 1) Compare Vmax from the double-reciprocal plot to the Vmax you estimated from the Michaelis-Menton curve. What are the problems with each kind of analysis? In which do you place more confidence, and why? Using the values from the Lineweaver-Burk plot ONLY: 1) Write out the Michaelis-Menton equation for this enzyme, using your calculated Vmax and Km. k) Calculate vo for [S] - 1.07 M 1) Is the enzyme saturated at 1.6 UM? How do you know? HINT: what is the maximal velocity this enzyme can attain? How close is the enzyme to maximal velocity at [S] -1.6 UM? Use a ratio and calculate the percentage of saturation

Answer 1

The plot of 1/V₀ versus 1/[S] can be drawn as follows.

0.00301 0.00251 30.0020 1N 0.0015 0.0010 11[S] (UM)

Equation of the line: 1/V₀ = 2.1109*10^-4 * 1/[S] + 8.29977*10^-4

Compare the above equation with the actual Line-Weaver Burk Plot equation, i.e. 1/V₀ = (K_M/V_max) * 1/[S] + 1/V_max

i.e. K_M/V_max = 2.1109*10^-4 s, and 1/V_max = 8.29977*10^-4 s/uM

i.e. The maximum rate, V_max = 1204.853 uM/s

And the Michaelis-Menten constant, K_M = 0.254 uM