Properties of bound trifluoroethanol complexes with horse liver alcohol dehydrogenase.

摘要: The substrate analogue 2,2,2-trifluoroethanol (TFE) has been used as a 19F NMR probe of the active site alcohol dehydrogenase from horse liver (LADH). We are able to directly observe single resonance assigned TFE in its ternary complex with LADH and nicotinamide adenine dinucleotide (NAD). chemical shift this is independent pH between values 6.2 8.9, suggesting that bound does not change ionization state range. Both by self-exchange measurements ligand-displacement studies pyrazole, we also find displacement NAD linear function proton concentration over similar range, more rapid desorption occurring at lower values. This suggests pK 6.4 for process seen previously Kvassman Pettersson [Kvassman, J., & Pettersson, G. (1980) Eur. J. Biochem. 103, 557] due TFE. These show lifetime quite long (400s) 8.7, use kinetic trapping reagent single-turnover stopped-flow experiments. Binding isotherms saturated 8.7 or pyrazole 7.5 reveal essentially no cooperative behavior. time courses described above all adequately fit first-order processes, thus giving evidence heterogeneity site-site interaction binding these ligands dimeric LADH. reagents further explore question point view following paper [Anderson, D. C., Dahlquist, F.W. (1982) Biochemistry (following issue)].