Employing deuterium kinetic isotope effects to uncover the mechanism of (R)-3-hydroxybutyrate dehydrogenase.

Abstract

Short-chain dehydrogenases/reductases (SDR) form a large enzyme superfamily playing important roles in health and disease. Furthermore, they are useful tools in biocatalysis. Unveiling the nature of the transition state for hydride transfer is a crucial undertaking toward defining the physicochemical underpinnings of catalysis by SDR enzymes, including possible contributions from quantum mechanical tunneling. Primary deuterium kinetic isotope effects can uncover the contribution from chemistry to the rate-limiting step and potentially provide detailed information on the hydride-transfer transition state in SDR-catalyzed reactions. For the latter, however, one needs to determine the intrinsic isotope effect: that which would be measured if hydride transfer were rate determining. Alas, as is the case for many other enzymatic reactions, those catalyzed by SDRs are often limited by the rate of isotope-insensitive steps, such as product release and conformational changes, which masks the expression of the intrinsic isotope effect. This can be overcome by the powerful yet underexplored method of Palfey and Fagan via which intrinsic kinetic isotope effects can be extracted from pre-steady-state kinetics data. SDRs are ideal systems to which this method can be applied. We have employed this approach to elucidate the transition states for hydride transfer catalyzed by NADH-dependent cold- and warm-adapted (R)-3-hydroxybutyrate dehydrogenase. Experimental conditions which simplify the analysis are discussed.