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

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.

Allosteric Inhibition of ATP Phosphoribosyltransferase by Protein:Dipeptide and Protein:Protein Interactions.

ATP phosphoribosyltransferase (ATPPRT) catalyzes the first step of histidine biosynthesis in bacteria, namely, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to generate -(5-phospho-β-d-ribosyl)-ATP (PRATP) and pyrophosphate. Catalytic (HisG) and regulatory (HisZ) subunits assemble in a hetero-octamer where HisZ activates HisG and mediates allosteric inhibition by histidine. In , HisG is necessary for the bacterium to persist in the lung during pneumonia.

Transition States for Psychrophilic and Mesophilic ()-3-Hydroxybutyrate Dehydrogenase-Catalyzed Hydride Transfer at Sub-zero Temperatures.

()-3-Hydroxybutyrate dehydrogenase (HBDH) catalyzes the NADH-dependent reduction of 3-oxocarboxylates to ()-3-hydroxycarboxylates. The active sites of a pair of cold- and warm-adapted HBDHs are identical except for a single residue, yet kinetics evaluated at -5, 0, and 5 °C show a much higher steady-state rate constant () for the cold-adapted than for the warm-adapted HBDH. Intriguingly, single-turnover rate constants () are strikingly similar between the two orthologues.

Dissecting the Mechanism of ()-3-Hydroxybutyrate Dehydrogenase by Kinetic Isotope Effects, Protein Crystallography, and Computational Chemistry.

The enzyme ()-3-hydroxybutyrate dehydrogenase (HBDH) catalyzes the enantioselective reduction of 3-oxocarboxylates to ()-3-hydroxycarboxylates, the monomeric precursors of biodegradable polyesters. Despite its application in asymmetric reduction, which prompted several engineering attempts of this enzyme, the order of chemical events in the active site, their contributions to limit the reaction rate, and interactions between the enzyme and non-native 3-oxocarboxylates have not been explored. Here, a combination of kinetic isotope effects, protein crystallography, and quantum mechanics/molecular mechanics (QM/MM) calculations were employed to dissect the HBDH mechanism.

Linear Eyring Plots Conceal a Change in the Rate-Limiting Step in an Enzyme Reaction.

The temperature dependence of psychrophilic and mesophilic ( R)-3-hydroxybutyrate dehydrogenase steady-state rates yields nonlinear and linear Eyring plots, respectively. Solvent viscosity effects and multiple- and single-turnover pre-steady-state kinetics demonstrate that while product release is rate-limiting at high temperatures for the psychrophilic enzyme, either interconversion between enzyme-substrate and enzyme-product complexes or a step prior to it limits the rate at low temperatures. Unexpectedly, a similar change in the rate-limiting step is observed with the mesophilic enzyme, where a step prior to chemistry becomes rate-limiting at low temperatures.