Interpretation of transient-state kinetic isotope effects.
In contrast to steady-state kinetic isotope effects (KIE's), transient-state KIE's are dependent on both time and signal source. We developed a theory which predicts the behavior of transient-state KIE's, permits the calculation of the intrinsic KIE, and makes possible the assignment of various optical signals to either pre- or post-hydride transfer events. We proved that the behavior of KIEobs for a reversible two-step reaction for all possible values of the rate constants and all possible ratios of intermediate and product contributions obeys three simple rules (assuming that the isotope-sensitive step involves a hydride transfer): (1) If only the post-hydride species contributes to the observed signal, KIEobs = KIEint at t = 0 and then decreases with time. (2) If only the pre-hydride species contributes to the observed signal, then KIEobs = 1 at t = 0 and then decreases with time. (3) If both pre- and post-hydride species contribute to the observed signal, then KIEobs = 1 at t = 0 and then will either rise or fall with time depending on the relative molar signal coefficients of the pre- and post-hydride species. We provide experimental evidence that the phenomena predicted by this theory do in fact occur in enzyme-catalyzed reactions.
Fisher
H F
HF
Department of Biochemistry, University of Kansas Medical Center, Missouri, USA.
Saha
S K
SK
D007553:Isotope Labeling; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D013997:Time Factors