Direct calorimetric analysis of the enzymatic activity of yeast cytochrome c oxidase.
The kinetic and thermodynamic parameters associated with the enzymatic reaction of yeast cytochrome c oxidase with its biological substrate, ferrocytochrome c, have been measured by using a titration microcalorimeter to monitor directly the rate of heat production or absorption as a function of time. This technique has allowed determination of both the energetics and the kinetics of the reaction under a variety of conditions within a single experiment. Experiments performed in buffer systems of varying ionization enthalpies allow determination of the net number of protons absorbed or released during the course of the reaction. For cytochrome c oxidase the intrinsic enthalpy of reaction was determined to be -16.5 kcal/mol with one (0.96) proton consumed for each ferrocytochrome c molecule oxidized. Activity measurements at salt concentrations ranging from 0 to 200 mM KCl in the presence of 10 mM potassium phosphate, pH 7.40, and 0.5 mM EDTA display a biphasic dependence of the electron transferase activity upon ionic strength with a peak activity observed near 50 mM KCl. The ionic strength dependence was similar for both detergent-solubilized and membrane-reconstituted cytochrome c oxidase. Despite the large ionic strength dependence of the kinetic parameters, the enthalpy measured for the reaction was found to be independent of ionic strength. Additional experiments involving direct transfer of the enzyme from low to high salt conditions produced negligible enthalpy changes that remained constant within experimental error throughout the salt concentrations studied (0-200 mM KCl). These results indicate that the salt effect on the enzyme activity is of entropic origin and further suggest the absence of a major conformational change in the enzyme due to changes in ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)
Morin
P E
PE
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Freire
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D002152:Calorimetry, Differential Scanning; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry*; D004795:Enzyme Stability; D007700:Kinetics; D009994:Osmolar Concentration; D011522:Protons; D013816:Thermodynamics; D015003:Yeasts / Q000187:drug effects / Q000201:enzymology*
D011522:Protons; D003576:Electron Transport Complex IV