With these trailing mails, I tried to know the optimum pH for E1 enzyme.
What amount of E2 enzyme should I use ? If I use excess of E2 enzyme (~ 20 time of E1) I can see only a view for optimum pH. If I decrease the amount of E2 (~6-8 time of E1) I can see a cleared view of the pH profile. My question is: what amount of E2 enzyme should I use if I am using 3 microgram of E1 enzyme?? E2 enzyme has Km (for NADH) 17 microM and for P1 the Km is 15 microM and the optimum pH is 6.5. Thank you On Fri, May 8, 2015 at 8:21 PM, R. M. Garavito <rmgarav...@gmail.com> wrote: > 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: rmgarav...@gmail.com > <garav...@gmail.com>* > ****************************************************************** > > > > > On May 8, 2015, at 9:23 AM, rohit kumar <rohit...@gmail.com> 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 > > > -- WITH REGARDS Rohit Kumar Singh Lab. no. 430, P.I. Dr. S. Gourinath, School of Life Sciences, Jawaharlal Nehru University New Delhi -110067
