Abstract
The kinetic characteristics of the hydrogen uptake reaction of hydrogenase, obtained by conventional activity measurements, led to the proposal of an autocatalytic reaction step in the hydrogenase cycle or during the activation process. The autocatalytic behavior of an enzyme reaction may result in oscillating concentrations of enzyme intermediates and/or products contributing to the autocatalytic step. This behavior has been investigated in the early phase of the hydrogenase-methyl viologen reaction. To measure fast hydrogenase kinetics, flash-reduced methyl viologen has been used as a light-induced trigger in transient kinetic phenomena associated with intermolecular electron transfer to hydrogenase. Here we report fast kinetic measurements of the hydrogenase-methyl viologen reaction by use of the excimer laser flash-reduced redox dye. The results are evaluated on the assumption of an autocatalytic reaction in the hydrogenase kinetic cycle. The kinetic constants of the autocatalytic reaction, i.e. the methyl viologen binding to and release from hydrogenase, were determined, and limits of the kinetic constants relating to the intramolecular (intraenzyme) reactions were set.
Highlights
The enzymatic activity of hydrogenase is determined routinely, a number of contradictory results have been published
The results are evaluated on the assumption of an autocatalytic reaction in the hydrogenase kinetic cycle
The kinetic constants of the autocatalytic reaction, i.e. the methyl viologen binding to and release from hydrogenase, were determined, and limits of the kinetic constants relating to the intramolecular reactions were set
Summary
The enzymatic activity of hydrogenase is determined routinely, a number of contradictory results have been published. We report fast kinetic measurements of the hydrogenase-methyl viologen reaction by use of the excimer laser flash-reduced redox dye.
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