Take Home Exam 2

1)Suppose that the data below are obtained for an enzyme-catalyzed reaction in the presence and absence of inhibitor Z.
    a) Using  double reciprocal plots of the data (Lineweaver-Burk), determine the type of inhibition that has occurred. Include your plots and any calcuations made.
    b) Calculate the inhibition constant, K_i for substance Z, assuming that the concentration of Z in the mixture is 0.20 mM. Show calculations.
 

[S],mM	v(mmol mL-1 min-1)	v(mmol mL-1 min-1)
	Without Z	With Z
0.20	5.0	3.0
0.40	7.5	5.0
0.80	10.0	7.5
1.0	10.7	8.3
2.0	12.5	10.7
4.0	13.6	12.5

2) Although the Lineweaver-Burk plot is the most widely used plotting form for enzyme kinetic data, it suffers a major disadvantage. If linear increments of [S] are used, thereby minimizing measurement errors in the laboratory, data points will be obtained that cluster near the vertical axis in an L-B plot. Thus the intercept of the ordinate can be determined with great accuracy, but the slope of the line will be subject to considerable error, because the least reliable points, those obtained at low substrate concentration, have greater weight in establishing the slope.

Because of the limitation of L-B plots, other linear plots have been devised. The E-H plot, (v  vs. v/[S]) is perhaps the most useful.

a) Rearrange and manipulate the M-M equation to give v as a function of (v/[S] )(i.e. , v = ???). Put it in the slope-intercept form.
b)What is the significance of the 1) slope, 2) the vertical (y) intercept and 3) the horizontal (x) intercept in a plot of v versus v/[S] ?
c) Data below were obtained for the hydrolysis of o-nitrophenyl-b-D-galactoside (ONPG) by E. coli beta-galactosidase. Use both a Lineweaver-Burk plot and a E-H plot to determine Km and Vmax. How do they compare? Include both plots. I suggest you graph them in Excel or something similar and obtain a linear regression fit.  Show your calculations.
 

[S],mM	v(mmol mL-1 min-1)
0.50	8.93
1.0	14.29
1.5	16.52
2.0	19.20
2.5	19.64