Abstract
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. This observation may have implications for past and future interpretations of temperature–rate profiles.
Highlights
Temperature−rate profiles are powerful tools for gaining insight into thermodynamic activation parameters of chemical reactions.[1]
Plotting ln(k/T) versus 1/T (Eyring plot) often results in a straight line, and fitting data to eq 1 yields the activation enthalpy and entropy, assuming they are constant over the temperature range and recrossing is negligible.[1,6,7]
Nonlinear Eyring plots have been reported,[8] and a recent and elegant hypothesis for interpreting them invokes a role for activation heat capacity in enzyme catalysis upon fitting data to eq 2.9,10 In eqs 1 and 2, k is the rate constant; kB, h, and R are the Boltzmann, Planck, and gas constants, respectively; ΔH⧧ and ΔS⧧ are the activation enthalpy and entropy, respectively; T is the temperature; T0 is a reference temperature; ΔH⧧T0 and ΔS⧧T0 are ΔH⧧ and ΔS⧧ at T0, respectively; and ΔCp⧧ is the activation heat capacity
Summary
Temperature−rate profiles are powerful tools for gaining insight into thermodynamic activation parameters of chemical reactions.[1]. The solvent viscosity effects on kcat suggest that the curvatures in the Eyring plots and the consequent negative ΔCp⧧ calculated for PaHBDH may report on distinct steps when acetoacetate and 3-oxovalerate are used as substrates.
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