Substitution of light isotopes for their heavy counterparts frequently leads to changes in the observed reaction rates for chemical reactions in which the isotope is involved. The measurement of such kinetic isotope effects is a very powerful tool for the diagnosis of reaction mechanism.
For example, when a proton (protium) is replaced by deuterium or tritium, changes in the rate of the reaction as large as several fold may take place. However, such substitutions can also yield small changes (10 - 20 %) in the rate, requiring specialized techniques to define their magnitude. Larger effects usually result when the isotope is transferred in the transition state that limits the overall rate of the reaction. Such reactions give what are referred to as primary kinetic isotope effects (PKIE's).
Isotope effects are primarily due to differences in the zero point energies (ZPE) between the light and heavy atoms. The figure below indicates the potential energy well for vibrations of the light and heavy atoms, in the reactant (R ), transition (T) and product (P) states. The difference in the isotopically sensitive vibrational frequencies between the light and heavy atoms in the reactants is the major contributor to the isotope effect. This energy difference results in the light reacting faster than the heavy isotope.
Kinetic isotope effects are measured in order to obtain information on either 1) the mechanistic sequence of a multi-step chemical reaction, or 2) the transition state structure of a simple reaction. Enzymes are highly efficient catalysts due to their ability to bind very tightly to and thus stabilize transition state structures. They generally catalyze chemical reactions via a multi-step sequence. Therefore, KIE's are extremely useful tools for the analysis of enzyme mechanisms since they can provide information on both the nature of the rate-limiting step(s) and the transition state structure of the rate-limiting step, if it is sensitive to isotopic substitution.
Multiple kinetic isotope effects provide a means for determining if a reaction takes place in a concerted or stepwise fashion. If one studies a system with two isotopically sensitive steps and observes KIE's on the two steps individually, it may be possible to diagnose if the reaction is stepwise or concerted based on the changes in one KIE due to introduction of the second isotope. This is illustrated in the following figure. For a reaction with multiple partially rate determining steps, one wants to know if two protons are transferred in a single step (concerted), or if a single proton is transferred in two steps (stepwise).
In this example, the KIE at position 1 is actually being measured, while we can introduce a proton or deuteron at position 2. If the two isotopically sensitive steps take place on separate steps, one will see a reduction in the KIE at position 1, because deuteration at position 2 makes it slower and thus more rate determining. On the other hand, if both proton transfers occur on the same step, this step become more rate determining for the overall reaction, thus resulting in an increase in the KIE at position 1. This technique represents a powerful tool for elucidating the detailed mechanistic chemistry of enzyme catalyzed reactions.