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• W3175879886 abstract "(R)-3-Hydroxybutyrate dehydrogenase (HBDH) catalyzes the NADH-dependent reduction of 3-oxocarboxylates to (R)-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 (kcat) for the cold-adapted than for the warm-adapted HBDH. Intriguingly, single-turnover rate constants (kSTO) are strikingly similar between the two orthologues. Psychrophilic HBDH primary deuterium kinetic isotope effects on kcat (Dkcat) and kSTO (DkSTO) decrease at lower temperatures, suggesting more efficient hydride transfer relative to other steps as the temperature decreases. However, mesophilic HBDH Dkcat and DkSTO are generally temperature-independent. The DkSTO data allowed calculation of intrinsic primary deuterium kinetic isotope effects. Intrinsic isotope effects of 4.2 and 3.9 for cold- and warm-adapted HBDH, respectively, at 5 °C, supported by quantum mechanics/molecular mechanics calculations, point to a late transition state for both orthologues. Conversely, intrinsic isotope effects of 5.7 and 3.1 for cold- and warm-adapted HBDH, respectively, at -5 °C indicate the transition state becomes nearly symmetric for the psychrophilic enzyme, but more asymmetric for the mesophilic enzyme. His-to-Asn and Asn-to-His mutations in the psychrophilic and mesophilic HBDH active sites, respectively, swap the single active-site position where these orthologues diverge. At 5 °C, the His-to-Asn mutation in psychrophilic HBDH decreases Dkcat to 3.1, suggesting a decrease in transition-state symmetry, while the His-to-Asn mutation in mesophilic HBDH increases Dkcat to 4.4, indicating an increase in transition-state symmetry. Hence, temperature adaptation and a single divergent active-site residue may influence transition-state geometry in HBDHs." @default.
• W3175879886 created "2021-07-05" @default.
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• W3175879886 date "2021-06-30" @default.
• W3175879886 modified "2023-10-17" @default.
• W3175879886 title "Transition States for Psychrophilic and Mesophilic (<i>R</i>)-3-Hydroxybutyrate Dehydrogenase-Catalyzed Hydride Transfer at Sub-zero Temperatures" @default.
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• W3175879886 doi "https://doi.org/10.1021/acs.biochem.1c00322" @default.
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