Mechanism from isotope effects

Isotope effects represent perhaps one of the most versatile tools available to investigators interested in the determination of reaction mechanism, pa rticularly in the case of the mechanistic enzymologist. Interpretation of i sotope effect data is somewhat more difficult for enzyme reactions, since t he chemical or isotope-dependent step(s) is(are) normally not solely rate-l imiting as they are for non-enzyme-catalyzed reactions. One can, however, t ake advantage of rate-limitation by multiple steps in an enzyme-catalyzed r eaction to obtain information on a number of aspects of mechanism. In this paper, simple theory for the application of isotope effects to reaction mec hanism is developed, and applied to organic reactions and those catalyzed b y enzymes. Techniques used to measure isotope effects depend somewhat on th e isotope used, that is radioisotope vs, stable isotope, or hydrogen isotop e vs. heavier atoms. Techniques to be discussed include competitive and non competitive (or internal discrimination) measurements. In enzyme-catalyzed reactions, information can be obtained on the order of addition of reactant s and release of products, and this will be illustrated using the 6-phospho gluconate and alcohol dehydrogenase reactions. The use of multiple isotope effects can be used to distinguish between stepwise and concerted reactions , and this will be illustrated with the formate and glucose 6-phosphate deh ydrogenase and melic enzyme reactions.