The Arrhenius activation energy has to do with the energetics of the catalytic reaction, whereas thermostability has to do with the energetics of protein folding. Therefore, it would be incorrect to calculate Arrhenius activation energy from a thermostability study. Moreover, the range of temperatures chosen over which to make the Arrhenius plot must not be so high that the protein begins tolose activity due to denaturation.

The Arrhenius plot is log(kcat) versus 1/T. In order to determine kcat, you must measure Vmax at each temperature, not just V at a fixed concentration of substrate. Since the Km is also affected by temperature, there is the possibility that when you change the temperature the enzyme will not be saturated with substrate, giving a false low value for Vmax. Therefore, best practice would be to make a Michaelis plot (V versus [S]) at each temperature. The enzyme concentration does not have to be kept constant because you divide Vmax by [E] in order to get kcat.

S (substrate) should  be constant and saturated in all series. Do not worried about that. Just incubate the enzyme at the desired temperature and buffer without substrate and take aliquots to measure residual activity at several times. You have to draw a log plot of residual activity versus time at different times. The slopes of those lines are proportional to the inactivation rates of the enzyme at every temperature. Then,, you have to final plot those slopes against 1/ºK, and the new slope is proportional to Ea. Good luck

Thank u for replying me so soon.

I kept [S] and [E] concentration constant throughout all temp experiments....

The Arrhenius activation energy has to do with the energetics of the catalytic reaction, whereas thermostability has to do with the energetics of protein folding. Therefore, it would be incorrect to calculate Arrhenius activation energy from a thermostability study. Moreover, the range of temperatures chosen over which to make the Arrhenius plot must not be so high that the protein begins tolose activity due to denaturation.

The Arrhenius plot is log(kcat) versus 1/T. In order to determine kcat, you must measure Vmax at each temperature, not just V at a fixed concentration of substrate. Since the Km is also affected by temperature, there is the possibility that when you change the temperature the enzyme will not be saturated with substrate, giving a false low value for Vmax. Therefore, best practice would be to make a Michaelis plot (V versus [S]) at each temperature. The enzyme concentration does not have to be kept constant because you divide Vmax by [E] in order to get kcat.