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

• Published in: Biochemistry (Easton) 2018-12, Vol.57 (49), p.6757-6761
• Main Authors: Machado, Teresa F. G, Gloster, Tracey M, da Silva, Rafael G
• Format: Article
• Language: English
• Subjects: Acetoacetates - metabolism; Acinetobacter baumannii - enzymology; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Biocatalysis; Communication; Hydroxybutyrate Dehydrogenase - chemistry; Hydroxybutyrate Dehydrogenase - metabolism; Kinetics; Linear Models; Models, Biological; Psychrobacter - enzymology; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Solvents; Substrate Specificity; Temperature; Valerates - metabolism; Viscosity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.8b01099

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.