Fast kinetics analysis of the peroxidatic reaction catalysed by horse liver alcohol dehydrogenase.
The kinetics of the enzymatic step of the peroxidatic reaction between NAD and hydrogen peroxide, catalysed by horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1), has been investigated at pH 7 at high enzyme concentration. Under such conditions no burst phase has been observed, thus indicating that the rate-limiting step in the process, which converts NAD into Compound I, either precedes or coincides with the chemical step responsible for the observed spectroscopic change. Kinetic analysis of the data, performed according to a simplified reaction scheme suggests that the rate-limiting step is coincident with the spectroscopic (i.e., chemical) step itself. Furthermore, the absence of a proton burst phase indicates the proton release step does not precede the chemical step, in contrast with the case of ethanol oxidation. A kinetic effect of different premixing conditions on the reaction rate has been observed and attributed to the presence of NADH formed in the 'blank reaction' between NAD and residual ethanol tightly bound to alcohol dehydrogenase. A molecular mechanism for the enzymatic peroxidation step is finally proposed, exploiting the knowledge of the much better known reaction of ethanol oxidation. Inhibition of this reaction by NADH has been investigated with respect to H2O2 (noncompetitive, Ki about 10 microM) and to NAD (competitive, Ki about 0.7 microM). The effect of temperature on the steady-state reaction state (about 65 kJ/mol activation energy) has also been studied.
Favilla
R
R
Department of Physics, University of Parma, Italy.
Giordani
B
B
Mazzini
A
A
D000426:Alcohol Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008433:Mathematics; D008962:Models, Theoretical; D010544:Peroxidases; D013816:Thermodynamics
D000426:Alcohol Dehydrogenase; D010544:Peroxidases