Question

Prostaglandins are a class of eicosanoids, fatty acid derivatives with a variety of extremely potent actions on vertebrate tissues. They are responsible for producing fever and inflammation and its associated pain. Prostaglandins are derived from the 20-carbon fatty acid arachidonic acid in a reaction catalyzed by the enzyme prostaglandin endoperoxide synthase. This enzyme, a cyclooxygenase, uses oxygen to convert arachidonic acid to PGG2, the immediate precursor of many different prostaglandins. Arachidonic acid (MM) 0.245 0.294 Rate of formation of PGG2 (mM/min) 20.3 22.9 28.9 36.6 47.4 Rate of formation of PGG2 with 10 mg/ml ibuprofen (mM/min) 12.7 14.6 19.6 26.9 39.5 0.441 0.735 1.72 The kinetic data given in the table are for the reaction catalyzed by a mutant of prostaglandin endoperoxide synthase. Focusing here on the first two columns, determine the Vmax and Km of the enzyme. mM/min Vmax = mM Km =

Answer 1

1/V	1/S
0.04926108	4.08163265
0.04366812	3.40136054
0.03460208	2.2675737
0.0273224	1.36054422
0.02109705	0.58139535

Plot 1/V vs 1/S to fit it to the 1 / v = 1/Vmax + (Km/Vmax) *1 / [S]

We get 1/Vmax as the intercept which equals 0.0163964

Vmax = 60.988

Slope = Km / Vmax = 0.008036662 mM/min

Km = Vmax*Slope = 60.988*0.008036662 = 0.049 mM

Now similarly plot 1/V vs 1/(Inhibitor presence); you will notice no change in Vmax

If we plot the points (Column 1 and 3) for when the inhibitor ibuprofen is present we see the same y-intercept, the Vmax doesn't change if the inhibitor is there. This is indicative of a competitive inhibitor.