Name of page Secondary Plot

A secondary plot uses the intercept or slope from several Lineweaver-Burk plots to find additonal kinetic constants.

For example, when a set of v by [S] curves from an enzyme with a ping–pong mechanism (varying substrate A, fixed substrate B) are plotted in a Lineweaver–Burk plot, a set of parallel lines will be produced.

The following Michaelis–Menten equation relates the initial reaction rate v0 to the substrate concentrations [A] and [B]:

The y-intercept of this equation is equal to the following:

The y-intercept is determined at several different concentrations of substrate B. The y-intercept values are then plotted versus 1/[B] to determine the Michaelis constant for substrate B, , as shown in the Figure. The slope is equal to times and the intercept is equal to 1 over .

Secondary Plot of Enzyme System Horseradish Peroxidase and o-Phenylenediamine (with hydrogen peroxide as the second substrate)

Secondary Plot in Inhibition Studies

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A secondary plot may also be used to find a specific inhbition constant, kI.

For a competitive inhibitor, the apparent Michaelis constant is equal to the following:

 

The slope of the Lineweaver-Burk plot is therefore equal to:

 

If one creates a Secondary plot consisting of the slope values from several Lineweaver-Burk plots of varying inhibitor concentration [I], the competitive inhbition constant may be found. The slope of the secondary plot divided by the intercept is equal to 1/kI. This method allows one to find the kI constant, even when the Michaelis constant and vmax values are not known.

References

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  • A. Cornish-Bowden. Fundamentals of Enzyme kinetics, Rev. ed., Portland: London, England, (1995) pp. 30-37, 56-57.
  • The Horseradish Peroxidase/ o-Phenylenediamine (HRP/OPD) System Exhibits a Two-Step Mechanism. M. K. Tiama and T. M. Hamilton, Journal of Undergraduate Chemistry Research, 4, 1 (2005).
  • J. N. Rodriguez-Lopez, M. A. Gilabert, J. Tudela, R. N. F. Thorneley, and F. Garcia-Canovas. Biochemistry, 2000, 39, 13201-13209.