Voltammetric measurement of Michaelis-Menten kinetics for a protein in a lipid film reacting with a protein in solution

Rotating disc voltammetry data for the reaction of myoglobin, a model “enzyme” in a thin lipid film, with ferredoxin was shown to fit the Michaelis-Menten kinetic model, but did not fit a simple linear EC' model. Advantages of this thin-film method are a lipid bilayer environment similar to that of membrane bound enzymes, the tiny amounts of proteins required, and simplicity of instrumentation compared to alternative methodology. The apparent KM for ferredoxin reduction by reduced myoglobin was 112 μM, kcat was 102 s−1, and kcat/KM was 9.1 × 104 M−1 s−1 indicating relatively fast kinetics. Results suggest that this method should be amenable to kinetic studies of enzymes in lipid films with protein reaction partners in solution.