In these coupled enzyme assays, concentrations of [E1], [S], [E2] and
[NADH] need to be chosen such that the rate of the second reaction is at
least one to two orders of magnitude faster than the first, otherwise
the measured rate -d[NADH]/dt will not be rate-limited by -d[S]/dt.
Normally this is accomplished by using relatively high [E2] and [NADH]
and relatively low [E1]. When varying the pH of the reaction, care must
be taken to ensure that this condition is maintained, as the coupled
reaction (P1 -> P2) may also have a pH-rate variation. You can do a
crude pH-rate profile by choosing a single concentration of [S] and
assaying at various pH values to establish the approximate pH optimum.
However, to establish the true pH optima you would need to establish
values of kcat and kcat/Km at each pH value by measuring rates at
various [S] values and fitting to the Michaelis-Menten equation (if M-M
behavior is appropriate for E1).
Cheers,
_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: [email protected]
On 5/8/2015 9:23 AM, rohit kumar wrote:
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
