Liver Alcohol Dehydrogenase. II. Equilibrium Constants of Binary and Ternary Complexes of Enzyme, Coenzyme, and Caprate, Isobutyramide and Imidazole.

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Abstract
Using the fluorescence of the complexes ER and ERI the dissociation constants in 0.1 [mu] buffer, 23. 5[degree]C, at pH 7 of the caprate complexes Ke, i, Keo, I and KeI, O and of the isobutyramide complexes KE, I, KeR, I and KEI, R have been determined as well as KE, r, KE, O and the imidazole dissociation constants Ke, i, Ker, I, KEI, r, KeO, i and KEI, O for the pH range 6[long dash]9. Glycine used for buffering at pH 9[image], was found to give a binary but no ternary complex. In titrations in the presence of relatively high concentrations of alcohol, "unnatural" binary and ternary alcohol complexes were also found. KE, r was similar to previous values, but higher values obtained for KE, O resulted now in the disappearance of previous disagreements between the equilibrium and kinetic values at lower pH''s, the results now agreeing with the Theorell-Chance mechanism. Except for Ke, R, Ke, O and Keo, I the dissociation constants were essentially invarient with pH. The increase of Ke, R at high pH suggested it was probably sulphydryl groups which joined the protein to the DPNH ring. Analysis of the pH curve for KE, O indicated that of the free octahedral zinc bonds which in the free enzyme presumably hold water, one at higher pH (8.6) looses a proton. The resulting attraction between zinc-OH" and the positively charged pyridine ring of DPN, is considered responsible for the changes in Ke, O with pH.