Properties of bound trifluoroethanol complexes with horse liver alcohol dehydrogenase.
The substrate analogue 2,2,2-trifluoroethanol (TFE) has been used as a 19F NMR probe of the active site of alcohol dehydrogenase from horse liver (LADH). We are able to directly observe a single resonance assigned to TFE in its ternary complex with LADH and nicotinamide adenine dinucleotide (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 by 19F NMR self-exchange measurements and by ligand-displacement studies with pyrazole, we also find 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. This suggests that the pK of 6.4 for this process seen previously by Kvassman and Pettersson [Kvassman, J., & Pettersson, G. (1980) Eur. J. Biochem. 103, 557] is not due to the ionization of bound TFE. These studies also show that the bound lifetime of TFE in its ternary complex with LADH and NAD is quite long (400s) 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. TFE and pyrazole are used as reagents to further explore the question of site-site interaction from a kinetic point of view in the following paper [Anderson, D. C., & Dahlquist, F.W. (1982) Biochemistry (following paper in this issue)].
D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D000431:Ethanol / Q000031:analogs & derivatives*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D011485:Protein Binding; D014270:Trifluoroethanol / Q000494:pharmacology*
D000431:Ethanol; D014270:Trifluoroethanol; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase