School of Science and Technology Microbial and Enzyme Kinetics

Enzyme Kinetics Tutorial 3b Rate measurements were made on a food enzyme with and without the presence of an inhibitor with the following results Initial Rate, V0 / mol min-1 [S]/ M 25 50 75 100 150 200 Without inhibitor 8.20 12.86 15.51 17.95 20.02 22.64 With Inhibitor 5.65 8.16 9.68 11.04 13.56 15.71

Inhibitor concentration, [ I] = 20 M 1. 2. 3. Use either the Eadie-Hofstee or the Hanes plot to determine the type of inhibition Determine Km and V0 for the enzyme without inhibition and the inhibitor parameter, K I. What further tests would be necessary to improve the accuracy of estimate of KI? How would you calculate KI in this case?

EnzkinTut03b.doc

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GFW, 06/11/01

Rearrangement of Eadie-Hofstee and Hanes plots. The Eadie-Hofstee and Hanes plots may be rewritten to simplify them a. Eadie-Hofstee equation;

V0 Vmax V0 = [S ] K m K m
can be rearranged to give V0 = Vmax KmV0 [S ]

A graph of V0 (y-axis) vs V0 /[S] gives a straight line slope Km, intercept V0 . b. Hanes equation;

[S ] = [S ]
Vo

Vmax

Km Vmax

can be rearranged to give

[S ] = Vmax [S ] Km Vo

A graph of [S] (y-axis) vs. [S]/V0 gives a straight line slope, Vmax, intercept Km. Determining KI For systems where Km is affected. For systems where Vmax is affected

VO =

Vmax [S ] K MI + [S ]

V0 =

VMI [S ] K m + [S ]
Vmax 1 + [I ] K I

[I] where K MI = K m 1 + K I
rearranging gives
K MI [I ] = 1+ Km KI

where VMI =

rearranging gives

Vmax [I ] = 1+ V MI KI

A graph of KMI/Km will be a straight line, slope 1/KI and intercept 1. or KI may be calculated directly from
KI = [I] K MI K m 1

A graph of Vmax/VMI will give a straight line of slope 1/KI and intercept 1. or KI may be calculated directly from

KI =

VMI [I ] Vmax VMI

GFW, 06/11/01

EnzkinTut03b.doc

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