Dear Rohit, If you want to study E1 using the E2 reaction as a read-out, you have to make sure that the E1 reaction is rate-limiting and not the E2 reaction. Ideal would be if you could purchase P1 and first map the E2 reaction with P1. Once you know the Km, Vmax and pH behavior of E2, you can calculate how much E2 you should add to make sure the E2 reaction is not rate-limiting.
Alternatively, you could add varying amounts of E2 to the reaction mixture, to find out the point where the overall reaction speed no longer increases upon E2 addition, which indicates that the E1 reaction has become rate-limiting. As long as you do not know which of the two reactions is rate-limiting, it does not help to do the measurements at any specific pH. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von rohit kumar Gesendet: Freitag, 8. Mai 2015 15:23 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Enzyme kinetics Dear all, Sorry for off topic discussion. i have a doubt for the enzyme kinetic. [Inline image 2] Above is the reaction of my interest. it is a couple reaction. I want to determine the Km and Vmax for the E1 enzyme by the help of E2 enzyme by decreasing the amount of NADH (at 340 nm). if i don't know the optimum pH for E1. So is it ok, for publication point of view, to determine the Km and Vmax value of E1 enzyme at pH 7.5 ( a physiological pH) . Suppose if i determine the optimum pH of E1 by the help of E2 enzyme, that will be solely depend on the behaviour of E2 at different pH (if i am not wrong). Please suggest. Thanks in advance. -- WITH REGARDS Rohit Kumar Singh Lab. no. 430, P.I. Dr. S. Gourinath, School of Life Sciences, Jawaharlal Nehru University New Delhi -110067
