Chemical relaxation studies on the horse liver alcohol dehydrogenase system.
Chemical relaxation studies on the system horse liver alcohol dehydrogenase, nicotinamide adenine dinucleotide, and ethanol were conducted observing fluorescence changes between 400 and 500 nm. Temperature-jump experiments were performed at pH 6.5, 7.0, 8.0, and 9.0; concentration-jump experiments at pH 9.0. The reciprocal of the slowest relaxation time was found to be linearly dependent upon the enzyme concentration for relatively low enzyme concentrations, as predicted earlier. Use of the wide pH-range necessitated expression of the four apparent dissociation constants of the catalytic reaction cycle in terms of pH-independent constants. The system was described in terms of only one (or two) catalysis-linked protons not associated with the electron transfer. Protonic steps in a buffered system are in rapid equilibrium, too fast to be measured with the equipment available. Assuming only two of the four bimolecular reaction steps in the four-step cycle are fast compared to the remaining two, six cases may be considered with six expressions for the reciprocal of the slowest relaxation time. Comparison with the experimental data revealed that the bimolecular reaction steps governing the slowest relaxation time change with pH. Above the effective time resolution of the temperature-lump instrument with fluorescence detection (0.1 msec) only one other relaxation time was detectable and only at pH 9. This relaxation time, found to be independent of the concentration of all reactants within experimental error (r = 10 +/- 5 msec), is most likely due to an interconversion among ternary complexes.
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D002021:Buffers; D004579:Electron Transport; D000431:Ethanol / Q000378:metabolism; D006736:Horses; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D008433:Mathematics; D009243:NAD / Q000378:metabolism; D009991:Oscillometry; D009994:Osmolar Concentration; D013696:Temperature; D013997:Time Factors
D002021:Buffers; D009243:NAD; D000431:Ethanol; D000429:Alcohol Oxidoreductases