problem with km value determination

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mohammed's picture
problem with km value determination

I want to determine Km value for pfGST enzyme using CDNB as substrate. I know this substrate is very good for the enzyme. but when I increase the [glutathione] substrate the initial velocity still increase even if I exceed the kmX2. It looks not follow Michaelis-Menten equation.
Previous researchers use concentration range for glutathione 50-500  uM. to get Km=0.6mM
while I use conc  range of 0.1-5 mM to get km=3mM.