Abstract
Solvent isotope effects have long been used as a mechanistic tool for determining enzyme mechanisms. Most commonly, macroscopic rate constants such as kcat and kcat/Km are found to decrease when the reaction is performed in D2O for a variety of reasons including the transfer of protons. Under certain circumstances, these constants are found to increase, in what is termed an inverse solvent kinetic isotope effect (SKIE), which can be a diagnostic mechanistic feature. Generally, these phenomena can be attributed to an inverse solvent equilibrium isotope effect on a rapid equilibrium preceding the rate-limiting step(s). This review surveys inverse SKIEs in enzyme-catalyzed reactions by assessing their underlying origins in common mechanistic themes. Case studies for each category are presented, and the mechanistic implications are put into context. It is hoped that readers may find the illustrative examples valuable in planning and interpreting solvent isotope effect experiments.
Highlights
Solvent isotope effects (SIEs) can be a powerful tool for studying the mechanisms of enzyme-catalyzed reactions
As will be explained unlike normal SIEs, which can be the result of solvent kinetic isotope effects (SKIEs), solvent equilibrium isotope effects (SEIEs), or a combination of both, almost all inverse SIEs are dominated by a SEIE
The inverse SKIE was attributed to an SEIE arising from the rapid equilibrium of thiolate formation prior to binding of succinate, while the normal SKIE was attributed to the downstream transfer of a solvent-derived hydron, presumably involved in protonation of the carbonyl oxygen of glyoxylate
Summary
Solvent isotope effects (SIEs) can be a powerful tool for studying the mechanisms of enzyme-catalyzed reactions. SIEs report on solvent-sensitive steps in a mechanism—those steps that are involved or implicated in bond cleavage and bond formation of solvent-exchangeable hydrons as well as changes in the hydrogen bond network. This review focuses on inverse SIEs—that is, SIEs in which the reactions are favored in D2 O rather than H2 O—and offers several origins to aid in their mechanistic interpretation. The much more common normal SIEs (those favoring H2 O) have been discussed in great detail in the above reviews and elsewhere, but inverse SIEs have generally only received limited attention. Unlike normal SIEs, which can be the result of solvent kinetic isotope effects (SKIEs), solvent equilibrium isotope effects (SEIEs), or a combination of both, almost all inverse SIEs are dominated by a SEIE (i.e., an equilibrium that is more favorable in D2 O).
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