Effect of pH on the process of ternary-complex interconversion in the liver-alcohol-dehydrogenase reaction.
1. Kinetic relationships referring to multiple-turnover conditions have been derived for the slowest exponential transient appearing in two-substrate enzyme reactions proceeding by an ordered ternary-complex mechanism. The validity of these and previously derived theoretical relationships for this mechanism has been tested by application to the liver alcohol dehydrogenase reaction. 2. All essential features of the transient-state kinetics of alcohol oxidation by NAD+ in the liver alcohol dehydrogenase system can be qualitatively and quantitatively explained in view of the compulsory-order mechanism in the proposed scheme. There is no kinetic evidence for any half-of-the-sites reactivity of the enzyme. A consistent set of rate constants is reported for the enzymic oxidation of benzyl alcohol at pH 8.75. 3. Transient-state rate parameters for benzyl alcohol/benzaldehyde catalysis by liver alcohol dehydrogenase have been determined at different pH. The interpretation of such rate parameters is critically discussed with reference to their informative value for the purpose of determination of rate constants (k and k') for the process of ternary-complex interconversion in the proposed scheme. It is concluded that the apparent rate constant (k') for hydride transfer from benzyl alcohol to NAD+ is dependent on a proton dissociation step with a pKa of 6.4, whereas the rate constant (k) for hydride transfer from NADH to benzaldehyde exhibits no corresponding dependence on proton association. 4. The asymmetric pH dependence of the forward and reverse rate of ternary-complex interconversion during liver alcohol dehydrogenase catalysis appears to reflect an obligatory step of alcohol/alcoholate ion equilibration occurring at the ternary-complex level. It is suggested that the observed pKa 6.4 dependence of the transient rate of alcohol oxidation can be attributed to a coupled acid-base system involving minimally the enzyme-bound alcohol and the protein residues Ser-48 and His-51.
Effect of pH on the process of ternary-complex interconversion in the liver-alcohol-dehydrogenase reaction. 1. Kinetic relationships referring to multiple-turnover conditions have been derived for the slowest exponential transient appearing in two-substrate enzyme reactions proceeding by an ordered ternary-complex mechanism. The validity of these and previously derived theoretical relationships for this mechanism has been tested by application to the liver alcohol dehydrogenase reaction. 2. All essential features of the transient-state kinetics of alcohol oxidation by NAD+ in the liver alcohol dehydrogenase system can be qualitatively and quantitatively explained in view of the compulsory-order mechanism in the proposed scheme. There is no kinetic evidence for any half-of-the-sites reactivity of the enzyme. A consistent set of rate constants is reported for the enzymic oxidation of benzyl alcohol at pH 8.75. 3. Transient-state rate parameters for benzyl alcohol/benzaldehyde catalysis by liver alcohol dehydrogenase have been determined at different pH. The interpretation of such rate parameters is critically discussed with reference to their informative value for the purpose of determination of rate constants (k and k') for the process of ternary-complex interconversion in the proposed scheme. It is concluded that the apparent rate constant (k') for hydride transfer from benzyl alcohol to NAD+ is dependent on a proton dissociation step with a pKa of 6.4, whereas the rate constant (k) for hydride transfer from NADH to benzaldehyde exhibits no corresponding dependence on proton association. 4. The asymmetric pH dependence of the forward and reverse rate of ternary-complex interconversion during liver alcohol dehydrogenase catalysis appears to reflect an obligatory step of alcohol/alcoholate ion equilibration occurring at the ternary-complex level. It is suggested that the observed pKa 6.4 dependence of the transient rate of alcohol oxidation can be attributed to a coupled acid-base system involving minimally the enzyme-bound alcohol and the protein residues Ser-48 and His-51.