PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8KineticIsotopeEffectsIsotope effects on reaction catalyzed by G6P dehydrogenase.• Note difference in observed vs. intrinsic primary 2H isotope effect consistent with rate limiting character of H transfer from G6P C-1.• Note also that observed is much smaller than Dk, reflecting the effects of commitments.• Finally note D2O effects! Commitments double! This shows that the major effects of solvent change are on the rate constants for enzyme conformational changes that precede or follow the chemical step. This is typical for enzymes!Hermes et al. (1984) “Variation of transition state structure as a function of nucleotide in reactions catalyzed by dehydrogenases. 2. Formate Dehydrogenase”. Biochem, 23, 5479-88.Yeast Formate Dehydrogenase• Ordered Sequential BiBi kinetic mechanism, with NAD+ adding before formate.• Hydride transfer is completely rate-limiting in this enzyme’s mechanism, so intrinsic isotope effects are fully expressed.• This is an ideal system for using isotope effects to probe transition state structure.NAD+ analogs studied and their effects on kinetic parametersNAD analogs with increasingly positive redox potential shift primary deuterium isotope effect to higher values.Conversely, secondary deuterium isotope effects, with D on C-4 of NAD+, go down. Primary 13C isotope effects also go down.With increasingly positive redox potential of NAD+ analog, the transition state state occurs earlier. NAD+ occurs late, while acetylpyridine has a nearly symmetrical transition state.The CO2 analog azide binds to FDH 10,000x better than the formate analog nitrate, but the ratio is insensitive to nucleotide redox potential. Thus not the nature of the transition state but the shape of the initial binding pocket for formate is determining the rel. affinity.The change in transition state structure as the nucleotide is altered is therefore the result of redox difference, rather than any differences in binding