Liver Alcohol Dehydrogenase. III. Kinetics in the Presence of Caprate, Isobutyramide and Imidazole.

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Abstract
The effects on the kinetics of the LADH-coenzyme-substrate system of caprate and isobutyramide at pH 7 are described. Caprate and isobutyramide have only inhibitory actions, which were localized to different parts of the reaction cycle and used for the calculations of various velocity constants. The results were in agreement with the equilibrium data from Part II. Likewise, the effects of imidazole at pH 7 and 9 were studied in detail. It was found that imidazole could either increase (5[long dash]10 fold) or decrease the reaction velocity depending on the concentration of coenzymes and substrates. These effects were explained from the properties of the ternary enzyme-coenzyme-imidazole complexes. It was found to be a general rule that the attachment of substrates or competing inhibitors to the substrate binding sites od not interfere appreciably with the "on" velocity constants of the coenzymes. The sometimes very large changes in dissociation constants, observed in equilibrium measurements, were parallelled by changes in the "off velocity constants. The mode of attachment of coenzymes, substrates and inhibitors is discussed. Zinc seems to be bound to the protein by three out of six octahedral bonds. The free bonds presumably hold water molecules, one of which at higher pH (pK 8.60) looses a proton. The resulting attraction between Zn[long dash]OH- and the positively charged pyridine ring of DPN is held responsible for the changes in Ke, o with pH. This does not operate in the case of Ke, R where the pyridine ring carries no charge. It is concluded that in both ER and EO, the adenine moieties are attached to Zn by two bonds as third bidentate ligands, which as in the case of ethylene diamine are weak. Zinc is implicated as the center of a ternary complex with three bonds to the protein, two to the adenine and one to the substrates. In the binary complexes this latter bond is in EO joined to the pyridinium ring (Zn[long dash] OH-[long dash]N+) but not in ER when it is occupied by a water molecule or at high pH a hydroxo group. The two coenzymes select with great preference their partners by switching during interconversion to free one or the other of two out of three bonds, while each imposes by its different nature different properties on the third bond. An abortive ternary complex ERalc formed at high alcohol concentrations is more stable than the binary ER complex. This explains the inhibition of reaction velocity observed at high concentrations of alcohol in the LADH system. Glycine like ortho-phenanthroline and[alpha][alpha][image]-dipyridyl forms a binary complex with LADH but no ternary complexes including the coenzymes. It is assumed that glycine acts as a third bidentate ligand, excluding the adenine moieties of the coenzymes from being bound to the zinc.