Question

Question 3: The HIV protease is manufactured by the virus to cleave the polyprotein into functional parts of the virion. The velocity of the HIV protease with a viral protein target without inhibitor (column 1) and with a low concentration of a target inhibitory drug (column 2), shown below. The inhibitor was made by chemically modifying the viral protein target. Sphingosine (UM) V. (mg 'min) with no inhibitor ([U] = 0) 5.00E-06 1.00E-05 2.00E-05 4.00E-05 8.00E-05 1.60E-04 4.00E-05 5.33E-05 6.40E-05 7.11E-05 7.53E-05 7.76E-05 V. (mgmin) with low conc of inhibitor (LU) is low) 1.33E-05 2.29E-05 3.56E-05 4.92E-05 6.10E-05 6.92E-05 a) Before you start, what kind of inhibition do you expect to see, and why? b) Construct a Lineweaver-Burk plot, be clear which line is uninhibited, and which has inhibitor. c) Calculate Km and Vmax with and without inhibitor d) Sketch a line on the graph that represents what you might see if you ran the experiment again with even higher concentration of inhibitor ([U] is high). e) What kind of inhibitor is the modified peptide? f) How do you know? g) What does this mean in terms of where the inhibitor binds? h) Compare your hypothesis with the result i) On your plot, sketch what you would expect to see if you used a higher concentration of inhibitor. For practice, sketch (separate graphs please) what you would have expected to see if this inhibitor had worked in either of the two other common ways. (e.g., if you find the inhibitor is uncompetitive, sketch what you would expect for noncompetitive and competitive).

Answer 1

Michaelis Menten equation is

v = Vmax / (1 + (Km/[S]))

Lineweaver burk plot is between 1/[S] on x-axis and 1/Vo on y-axis.

substrate(UM) velocity (mm/min) inhibitor 5.00E-06 4.00E-05 1.00E-05 5.33E-05 2.00E-05 6.40E-05 4.00E-05 7.11E-05 8.00E-05 7.53E-05 1.60E-04 7.76E-05 1.33E-05 2.29E-05 3.56E-05 4.92E-05 6.10E-05 6.92E-05 1/ 1 200000 100000 50000 25000 12500 6250 /V(UM/min) 1/Vinhb 25000 75187.97 18761.72608 43668.12 1 5625 28089.89 14064.69761 20325.2 13280.21248 16393.44 12886.59794 14450.87 Chart Title 80000 70000 60000 50000 40000 30000 20000 10000 y = 0.3133x + 12456 y = 0.0625x+12500 250000 50000 100000 150000 200000 - 1/VuM/min) 4-1/Vinho ......... Linear (1/V(UM/min)) ......... Linear (1/Vinhb)

Equation of this graph is y = 0.0625x + 12500

According to this equation, slope = 0.0625 and intercept = 12500

Also, intercept = 1/Vmax = 12500

Vmax = 0.00008 uM/min

Slope = Km/Vmax = 0.0625

So, Km = 0.0625 *Vmax = 0.0625 *12500 = 781.25 uM

Answer : Km = 781.25 uM

Vmax = 0.00008 uM/min

Since intercept is same only Km is changed hence this represent competitive inhibition