The competition plot: A kinetic method to assess whether an enzyme that catalyzes multiple reactions does so at a unique site

Enzymes often act on more than one substrate, and the question then arises as to whether this can be attributed to the existence of two different enzy mes that have not been separated or, more interesting, to the presence of t wo different active sites in the same enzyme. The competition plot is a kin etic method that allows us to test with little experimentation whether the two reactions occur at the same site or at different sites. It consists of making mixtures of the two substrates and plotting the total rate against a parameter p that defines the concentrations of the two substrates in terms of reference concentrations chosen to give the same rates at p = 0 and p = 1, i.e., when only one of the substrates is present. With a slight modific ation of the equations it can also be applied to enzymes that deviate from Michaelis-Menten kinetics. If the two substrates react at the same site, th e competition plot gives a horizontal straight line; i.e., the total rate i s independent of p. In contrast, if the two reactions occur at two separate and independent sites a curve with a maximum is obtained; separate reactio ns with cross-inhibition generate curves with either maxima or minima accor ding to whether the Michaelis constants of the two substrates are smaller o r larger than their inhibition constants in the other reactions. Strategies to avoid ambiguous results and to improve the sensitivity of the plot are described. A practical example is given to facilitate the experimental prot ocol for this plot. (C) 2001 academic press.