A method for extracting rate constants from initial rates of stopped-flow kinetic data: application to a physiological electron-transfer reaction.
The most commonly used methods for analysis of stopped-flow kinetic data require performing a series of measurements in which one reactant is varied at concentrations significantly greater than the concentration of the other reactant. For enzyme-catalysed reactions this may not be possible, because the dissociation constants for the enzyme-substrate complex are often of the same order of magnitude as the high concentrations of enzyme that must frequently be used in stopped-flow studies. An alternative method of data analysis is presented which allows the determination of microscopic rate constants from initial rates of stopped-flow kinetic data in which substrate is varied in a range of concentrations approximately the same as the enzyme. This method also provides a simple and accurate method for determining k4, the rate of the reverse reaction. This method has been used to describe a physiological electron transfer reaction between a quinoprotein, methylamine dehydrogenase, and a copper protein, amicyanin. At 20 degrees C, the rate of the electron-transfer reaction from methylamine dehydrogenase to amicyanin was 24 s-1, and the dissociation constant for complex-formation was 1.9 microM.
Brooks
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HB
Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216-4505.
Davidson
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VL
D001426:Bacterial Proteins / Q000737:chemistry; D004579:Electron Transport; D007700:Kinetics; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry; D010231:Paracoccus denitrificans / Q000201:enzymology
D001426:Bacterial Proteins; C017079:mauC protein, Methylobacterium extorquens; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors