Question

A student carries out the enzymatic conversion of ethanol to acetaldehyde (shown) and collects the kinetic data below.

CH₃CH₂OH + NAD⁺ à CH₃CHO + NADH

Ethanol (µL)	Initial Reaction Rate (µM/s)
0.078	0.065
0.156	0.163
0.313	0.213
0.625	0.285
1.250	0.454
2.50	0.51
5.00	0.47

*Construct a Michaelis-Menten plot of the results in Excel. Show a scatter plot only (don’t connect the dots).  Estimate K_m and V_mas (because Excel doesn’t curve fit to the M-M equation).

*Construct a Lineweaver-Burk plot of the results. Calculate values for K_m and V_mas.

*Sketch and discuss kinetic results that would results if a competitive inhibitor was added to the reaction.

Answer 1

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

Where V is the rate of reaction, [S] is the concentration of Ethanol.

The equation of the plot: 1/V = 1.02073*1/[S] + 1.43738 .......... Equation 1

The actual Lineweaver-Burk plot equation: 1/V = K_M/V_max*1/[S] + 1/V_max .......... Equation 2

Where K_M is the Michaelis-Menten constant, Vmax is the maximum rate

By comparing both the equations 1 and 2, you will get

1/V_max = 1.43738 s/M,

i.e. V_max = 695.7 nM/s

Note: 1 nM = 10^-9 M, 1 M = 10^-6 M

And K_M/V_max = 1.02073 L*s/M

i.e. K_M = 695.7 nM/s * 1.02073 L*s/M

i.e. K_M = 0.710 L