Question

ErmTR is an enzyme found in S. pyogenes strains that are resistant to erythromycin. While working in the lab one afternoon you discover a small molecule (C23) that inhibits the activity of ermTR. To assess the type of inhibitor you discovered, you set up reactions with 6 different concentrations of erythromycin and varying amounts of the C23 small molecule you discovered. To each reaction you add 2 x 10-12 mol of ermTR (total reaction volume: 15 mL) and measure the initial velocities, Vo, by spectrophotometry. You obtained the following data:

Initial Velocity Vo (micromoles/min)

erythromycin	no inhibitor	1 mM inhibitor	2 mM inhibitor
1	0.059	0.045	0.037
2	0.105	0.082	0.071
4	0.174	0.144	0.119
6	0.222	0.190	0.160
8	0.258	0.210	0.197
16	0.340	0.310	0.281

Display the data on a Lineweaver-Burk plot in order to extract the Km and Vmax values. You can
generate the plot by hand on the graph paper on the last page or use a program, such as Excel, to plot the data. Submit the plot you generate with your test answers.

b) What are the KM and Vmax values for each of the samples? Show your calculations and explain how you obtain the answer for the “no inhibitor” sample.

c) What type of inhibitor is C23? Explain your answer.

d) Calculate the turnover number and the specificity constant for the enzyme sample that lacks the inhibitor C23.

Answer 1