Kinetic distinction between rapid-equilibrium random and abortive ordered enzymatic mechanisms using alternative substrates or kinetic isotope effects.

Alternative substrates, such as those isotopically-labeled, which differ in their rate constants of catalysis but not in their rate constants of binding, generate identical values of V/Ka in ordered kinetic mechanisms of bireactant enzymes. This is shown to be true even for the rapid-equilibrium ordered mechanism in which an abortive complex between free enzyme and the second substrate is formed. In contrast, rapid-equilibrium random mechanisms have non-identical values for V/Ka. Consequently, the effect of alternative substrates or isotope effects on V/Ka provides a means to distinguish between these nearly identical kinetic mechanisms.