13] Isotope effects: Determination of enzyme transition state structure

Abstract

Publisher Summary Enzymologists wish to determine the transition state structures for enzymatic reactions for several reasons: (1) to compare the mechanisms of enzymatic and nonenzymatic reactions in order to understand better the factors involved in enzymatic catalysis and (2) “transition state analogs” being used for drugs, to know what structure should be mimicked. Isotope effects have been used for many years to study the nonenzymatic reactions. For many enzymatic reactions, the observed isotope effects can be reasonably interpreted once the intrinsic values are known on the chemical steps. The problem is that the chemical step (or steps) of an enzymatic reaction is often not totally rate limiting, so that one has to either find a way to make this step rate limiting (by changing substrate, changing pH, or mutating the enzyme) or find a way to calculate the intrinsic isotope effect. This chapter discusses some basic definitions, and then describes the more modern methods of measuring isotope effects and determining intrinsic isotope effects. After that, it gives examples of the determination of transition state structure and what these studies tell about enzymatic mechanisms.