Mechanistic deductions from isotope effects in multireactant enzyme mechanisms

Abstract

In an enzymatic mechanism with 2 or more substrates, comparison of the isotope effects on the Vmax and on the apparent V/K values when each substrate concentration is varied allows one to deduce the kinetic mechanism and obtain quantitative information on the relative rates at which substrates dissociate from the enzyme, as opposed to undergoing reaction to give products. Theory is also presented for using the effects of other reactants on the apparent isotope effects determined by the equilibrium perturbation method to determine the same information. With liver alcohol dehydrogenase, DPN is not released at an appreciable rate from the E-DPN-cyclohexanol complex, while cyclohexanol is released much more rapidly than it reacts to give products, so that the mechanism appears ordered. With DPNH and cyclohexanone, the reaction is random since DPNH can be released from the ternary complex at a finite rate. With yeast alcohol dehydrogenase, acetone when present, prevents DPNH release from the enzyme so that the mechanism appears ordered but 2-propanol and DPN are released at equal rates from E-DPN-2-propanol so that the reaction is random in this direction.