Estimate the Km and Vmax in the absence and in the presence of inhibitorsDetermine what type of inhibitors A and B are. Explain your answer. Enzyme kinetics was analyzed in the absence of inhibitors, as well as in the presence of Inhibitor A and Inhibitor B. Plot allof the data on one graph as Michaelis-Menten saturation curves (scatter plot with smooth lines). Then, answer the questionsbelow:v (mmol/min)no inhibitorv (mmol/min)inhibitor Av (mmol/min)inhibitor B[S]33.92.41.295.33.32.4127.853.924116.66.736127.28.557212.27.611.8

Michaelis menten constant, Km= It is the substrate concentration required to produce half maximum velocity. V= Vmax[S]/Km + [S].........(eq1) where, V = measured velocity, Vmax = maximum velocity, S = substrate concentration, Km = michaelis menten constant The reciprocal of equation 1 is given by lineweaver equation: 1/V = (Km/Vmax) × [1/S]+ 1/Vmax The above equation is similar to equation of straight line y =mx+c, m= Km/Vmax, C= 1/Vmax Given data: S v(mmol/min) no inhibitor v (mmol/min) inhibitor A v (mmol/min) inhibitor B 3 3.9 2.4 1.29 6 5.3 3.3 2.4 12 7.8 5 3.9 24 11 6.6 6.7 36 12 7.2 8.55 72 12.2 7.6 11.81. Take reciprocal of S and V. 1/s 1/v without inhibitor 1/v with inhibitor A 1/v with inhibitor B 0.33 0.25 0.41 0.77 0.16 0.188 0.30 0.41 0.083 0.12 0.2 0.25 0.041 0.09 0.15 0.14 0.027 0.083 0.13 0.11 0.013 0.081 0.13 0.08 Form the straight line equation for the three inhibitors: Straight line equation without inhibitor: Y = 0.533x + 0.074 This equation is comparable to line-weaver equation therfore m= Km/Vmax and C= 1/Vmax C = 0.074=1/vmax Vmax = 1/0.074 = 13.5 mmol/min m= 0.533= Km/Vmax Km/Vmax = 0.533 Km = 0.533 × 13.5 = 7.19 Straight line equation with inhibitor A: Y = 0.883x + 0.118 1/Vmax = 0.118 Vmax = 8.4 mmol/min Km/Vmax = 0.883 Km = 7.41 Straight line equation with inhibitor B: Y = 2.176x + 0.051 1/Vmax = 0.051 Vmax = 19.6 mmol/min Km/Vmax = 2.17 Km = 42.5 2. So, with inhibitor A Vmax is decreasing while Km is not significantly changed so it is NON-COMPETITIVE INHIBITOR. With inhibitor B Vmax is not significantly increasing while Km is increasing so it is a COMPETITIVE INHIBITOR. MICHAELIS MENTEN CURVE