Properties of bound trifluoroethanol complexes with horse liver alcohol dehydrogenase

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Abstract
The substrate analog 2,2,2-trifluoroethanol (TFE) was used as a 19F NMR probe of the active site of alcohol dehydrogenase from horse liver (LADH). A single resonance assigned to TFE can be directly observed in its ternary complex with LADH and (NAD). The chemical shift of this resonance is independent of pH between values of 6.2 and 8.9, suggesting that bound TFE does not change ionization state in this range. Both 19F NMR self-exchange measurements and lig-and-displacement studies with pyrazole, show that displacement of TFE from its ternary complex with NAD is a linear function of proton concentration over a similar pH range, with more rapid desorption occurring at lower pH values. The pK of 6.4 for this process seen previously by Kvassman and Pettersson (1980) is probably not due to the ionization of bound TFE. The bound lifetime of TFE in its ternary complex with LADH and NAD is quite long (400 s) at pH 8.7, suggesting the use of TFE as a kinetic trapping reagent in single-turnover stopped-flow experiments. Binding isotherms of NAD to LADH saturated with TFE at pH 8.7 or with pyrazole at pH 7.5 reveal essentially no cooperative behavior. The displacement time courses described above are all adequately fit as first-order processes, thus giving no evidence for site heterogeneity or site-site interaction in the binding of these ligands to dimeric LADH.