ABSTRACT: The previously observed puzzling effect of 1o isotope on 2o kinetic isotope effects (KIEs) in alcohol dehydrogenases (ADHs) were compared to similar investigations on model reactions in which 2-propanol is oxidized by six NAD+ models (carbocations) in acetonitrile. The observed “abnormal” α-H/D and β-2o CH3/CD3 KIEs and the 1o isotope effect on the 2o KIEs from both solution and enzymatic hydride transfer systems indicate H-tunneling. The latter effect largely traces to the D-transfer process, and is pronounced in solution with sterically hindered carbocations. The steric hindrance effect hinders the reorganization of 2o H/D more in the D-tunneling, which occurs at a shorter donor-acceptor distance (DAD) than in the H-tunneling reaction. The effect of 1o isotope on the β-2o KIEs is significant but that on the α-2o KIEs is not, suggesting that the reorganization/vibrations of the β-CH3 group is more readily hindered by steric effect than the α-H group. In ADHs, however, the more densely packed protein active site environment induced from the D-tunneling that requires a shorter DAD significantly hinders the α-type 2o H/D’s reorganization resulting in deflated 2o KIEs and hence the effect of 1o isotope on α-2o KIEs.

Source of the puzzling effect of the primary isotope on secondary kinetic isotope effects in alcohol dehydrogenases

249th National Meeting (2015)

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