Rohit, Yes, you are wise to worry about this coupled reaction for a number of reasons. Your question allows me to pull my head out of grading lab final exams on this very concept. I agree with Herman's and Roger's comments. I have a couple of other items to mention:
Luckily, NAD(H)-linked enzymes like dehydrogenases are generally very fast enzymes and are often used as coupling enzymes. However, NAD(H)-linked enzymes like dehydrogenases are often reversible, but they can have preferred directions of reaction. Moreover, the reaction is always pH sensitive because of the mechanism: P1 + NADH + H+ <=> P2 + NAD+ + H2O . Hence, by changing the pH you can impact the flow of the many, although not all, NAD(H)-linked enzymes like dehydrogenases. So, simply choosing an E2 because it can do the reaction is not a good justification, particularly if the reverse reaction is more highly preferred. You will need to do some control experiments. Fortunately, many NAD(H)-linked dehydrogenases are also well documented in the literature. Going in the direction you want (P1 + NADH + H+ <=> P2 + NAD+ + H2O) can also be problematic on technical grounds. The E2 enzyme should be saturated with NADH (~10xKm) to ensure it is never rate-limiting due to limiting [NADH]. If E2's Km for NADH is greater than 50 micromolar, then the starting absorbance for the assay at 0.5 mM NADH (with 1 cm path length) will be ~3 AU. Sadly, the detectors on many spectrophotometers are not linear at these high absorbances and can give you bogus initial rate data. For the aspartate aminotransferase assay we do in our undergraduate lab (honed and optimized so they can all do it), we try to keep the initial absorbance to under 0.5 AU (or [NADH] is less than 0.08 mM). So test the linearity of your spectrophotometer; very few people do this. The old Varians and Beckman-Gilfords were very linear, but some CCD based spectrophotometers are crap. Bottom line, you also need to know the Km for NADH of E2 to see if you can do it on your spectrophotometer. Good luck, Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: [email protected] **************************************************************** On May 8, 2015, at 9:23 AM, rohit kumar <[email protected]> wrote: > Dear all, > > Sorry for off topic discussion. i have a doubt for the enzyme kinetic. > > > <image.png> > 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
