The phosphatase enzyme PTP1B catalyzes the removal of phosphate groups from spec

Question

The phosphatase enzyme PTP1B catalyzes the removal of phosphate groups from specific proteins and is involved in insulin signaling. Phosphatase inhibitors such an vanadate may be useful in the treatment of diabetes. The activity of PTP1B (at a concentration of 0.0SuM) was measured in the presence and absence of vanadate. The data is shown below: Without vanadate with vanadate 6.67 10 20 40 100 200 5.7 8.3 12.5 16.7 22.2 25.4 0.71 1.06 2.04 3.70 8.00 12.5 (a) Draw a Michaelis-Menten plot for the uninhibited enzyme. (b) Draw a Lineweaver-Burke plot for this enzyme with and without inhibitor (c) Determine KM and Vmax for this enzyme (d) Indicate in both graphs (a & b) where Vnan and Ksw can be recognized. (e) Calculate kcat. a) Michaelis-Menten plot

Explanation / Answer

Michaelis-Menten plot is V= VmaxS/(KM+S) , so a plot of V vs S need to be generated. Vmax is maxium rate . The location of Vmax is shown in the plot.

when V= Vmax/2, Vmax/2= Vmax*S(/KM+S), 2S= KM+S, KM=S, at V= Vmax/2, KM can be located.

Vmax is located by drawing a horizontal line at the point where V reaches saturation point.

Accordingly, Vmax( no inhibitor)= 27.5 nM/s , Vmax ( inhibitor)= 15 nM/s

at V= Vmax/2 , draw a horizontal line where ever it interesects the curve, draw a line downward to give S.

This is shown in the plot. from the plot , KM= 20 uM without inhibitor Km=100 ( with inhibitor)

Kcat= Vmax/ET= 27.5 *10-9 M/s /0.05*10-6 M=0.55/s, in the presence of inhibitor, KCat= 15*10-9/(0.05*10-6)= 0.3/s

2. Lineweaver berk plot is 1/V= (KM/Vmax)*1/S +1/Vmax

so a plot of 1/V vs 1/S gvies straight line whose slope is KM/Vmax and intercept is 1/Vmax . This is shown

this plot gives correct value of KM as well as Vmax

for no inhibition, from the plot intercept is 1/Vmax=0.035, Vmax= 1/0.035= 28.57 nM/s, KM= 28.57*0.911 =26.02 uM

for the case of inhibiton, Vmax = 28.57 same as that in the absence of inhibitor, KM= 28.57*9.132= 260.9 uM

Kcat= Vmax/ET= 28.57*10-9/ (0.05*10-6) /s =0.5714/s

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