Treatment of Enzyme Kinetic Data

The concerted two-state allosteric model proposed by Monod et al. (Monod, J., Wyman, J., and Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118) is extended to expressions representing the initial velocity as a function of substrate concentration for two-substrate enzymes for which random substrate binding and rapid equilibration of all binding steps prior to a rate-determining conversion in the ternary complex are assumed. It is shown that when the two different conformational states have different affinities for both subtrates, kinetic behavior which is different from that expected from simple analogy to the single substrate case may be obtained. The consequences of unequal intrinsic activities of the conformational states are also examined. The equations are further extended to include cases where the apparent Michaelis constant of one substrate depends on the concentration of the other substrate. It is shown that combined kinetic studies and equilibrium binding studies may be used to distinguish between some of the cases developed. A fundamental difference between the kinetic expressions and equilibrium binding equations for two-substrate allosteric enzymes is described. Under some of the conditions described, it is possible to obtain kinetic negative cooperativity with this model.