Steady-state kinetic analysis of mitochondrial respiratory enzymes from bovine heart mitochondria

Abstract

AbstractMitochondria is a decisive organelle of cells that produces adenosine triphosphate (ATP) by the process of oxidative phosphorylation of the Krebs cycle and the electron transport chain. The electron transport chain system of mitochondria embodies multiple enzyme supercomplexes including complex I to V which located in the inner membrane. Although the simple enzyme activity of some as-isolated complex has been studied so far, the steady-state kinetic analysis of each complex within the form of mitochondrial supercomplex has not been studied in depth. To this end, kinetic parameters of mitochondrial complex I–IV were determined using steady-kinetic analysis using corresponding substrates of them. Catalytic activity and binding affinity between substrates and enzymes were obtained by fitting the data to the Michaelis–Menten equation. Acquired kinetic parameters represented distinctive values depending on the complexes that can be interpreted by the characteristics of the enzymes including the distinction of substrates or the ratio of the enzyme itself under the supercomplex form. The indirect kcat of the mitochondrial enzymes were varied from 0.0609 to 0.334 s−1 in order of complex III, II, I, and IV and Km of substrates were also diverse from 5.1 μM to 12.14 mM. This is the first attempt to get exact kinetic values that should provide profound information to evaluate the mitochondrial function practically in advance.