I am working on an enzyme and calculating the amount of product formed with GC-MS method. So I have calculated the amount of product frormed in uM from the area under curve of product and known standards. I got a linear curve (product conc vs substrate conc) but I need help for how to calculate the Km and Kcat values from this graph.
1. Should I change the values to fit into lineweaver burk plot(as it is already straight line curve) or directly measure and how should I do that.
2. Also, i use internal standard only. Do you think it is enough to calculate exact conc or should I use external standards as well.
Thankyou in advance. That will be really helpful.
If your Michaelis-Menten curve (enzyme rate vs substrate concentration) is a straight line you are either too far above or too far below Km and need to increase the range of substrate concentrations you are testing. A linear series cannot be fitted to the Michaelis-Menten equation (which describes a section of a rectangular hyperbola) to give you meaningful kinetic parameters.
For quantification, external standards (ideally with internal standard to correction for analytical variation) will be more accurate, but for a given experiment single point internal calibration may be adequate. In terms of an enzyme kinetics experiment where your product concentration may vary across several orders of magnitude I would at least make sure the internal standard response is linear across the range of product you are quantifying.
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