The measurement of kinetic isotope effects (KIEs) has proved useful in many mechanistic studies of enzyme activity, most notably in enzyme-catalyzed hydrogen-transfer reactions. Primary KIEs (1° KIE) greater than unity indicate that transfer of the hydrogen species of interest is partially or fully rate limiting, and studies of the magnitude of the temperature and pressure dependence of these KIEs can inform on the mechanism of transfer. For example, KIE measurements have proved crucial in understanding the role of quantum mechanical tunneling in enzyme systems. The measurement of secondary KIEs (2° KIEs) is also informative and can be used to infer a significant tunneling contribution and details of transition state geometry. Here the deuterium label is introduced next to that of the transferred hydrogen. Measurements of 1° and 2° KIEs are being used increasingly in studies of H-transfer in flavoprotein enzymes and this requires the preparation of high purity and stereospecific labeled isotopologues. Strategies for the synthesis of labeled substrates are dependent on the enzyme system being studied. However, the nicotinamide coenzymes are often used in studies of flavoprotein enzyme mechanisms. Here, we provide practical details for the enzymatic synthesis of high purity deuterated isotopologues of the common biological coenzymes NADH and NADPH as well as the corresponding nonreactive mimics, tetrahydroNAD(P)H. Both forms of the coenzyme have proven useful in the study of mechanisms, particularly in relation to the involvement of quantum mechanical tunneling and dynamics in enzymatic H-transfer chemistry. The focus here is on practical considerations in the synthesis of these compounds. We also provide an abbreviated description of how measurements of KIEs can inform on flavoprotein mechanisms. The aim of this contribution is not to give a detailed description of the underlying theory (which has been reviewed extensively in the literature), but to provide a basic introduction and practical considerations for nonexpert readers who wish to incorporate such measurements in studies of enzyme mechanisms.
|Name||Methods in Molecular Biology|