Kinetics of the two-sited enzyme: II. A method of distinguishing between anticooperative and independent active sites based on competitive inhibition

Abstract

The presence of two active sites on an enzyme leads to downwardly curving Lineweaver-Burk plots if (A) the sites are independent, but have different Michaelis constants, or (B) if the sites interact anticooperatively to impair binding, but not catalysis, at the second site filled. Cases A and B are kinetically indistinguishable when only enzyme and substrate are present. However, equations derived by the rapid-equilibrium treatment show that the two cases have different patterns of competitive inhibition and become distinguishable in the presence of a suitable inhibitor. The inhibitor may decrease or increase the curvature of Lineweaver-Burk plots, but certain patterns have diagnostic value because they can occur only in case (B). In one type of diagnostic pattern, high concentrations of inhibitor cause the Lineweaver-Burk plots to curve upward, and cause the corresponding saturation curves to become sigmoid. The effect of the inhibitor is thus to make sites which are anticooperative appear to be cooperative. This suggests that the mere occurrence of sigmoid saturation curves is not necessarily evidence of cooperative binding effects, and may have uncertain significance in considerations of enzyme regulation.