I have recently started to read about metabolic control analysis.
I was very happy to know that there is a theoretical treatment
of the effects of enzyme amounts on "in vivo" pathway fluxes. I
work in the area of phytochemistry, more precisely with the
regulation of terpenoid indole alkaloid accumulation in plant
cell suspension cultures. I do not know any articles concerning
plant secondary metabolites which use m.c.a., do you? I have read
the original article by H. Kacser and J.A. Burns and am gaining
further insight reading more recent papers. Most of them concern
theoretical simulations and animal or microorganism biochemistry.
The papers on plant biochemistry that I have been able to find
deal with primary metabolism (starch and sugar accumulation).
I am trying to apply mca to my own experiments but am having some
difficulties. My question is the following:
Which enzyme(s) of the biosynthetic pathway limit the
biosynthesis of the product of my interest or
which enzyme(s) have the highest flux control coefficient?
Before knowing of the existence of mca I made the following
experiment to try to answer part of this question:
I compared the alkaloid accumulation and the activities of
enzymes of its biosynthetic pathway of cells growing under normal
culture conditions with cells to which a substrate of the pathway
had been added. This was done for two cell lines differing in
their alkaloid accumulation capacity.
In a previous experiment I had found that the differences in the
alkaloid accumulation of the two cell lines was due to
differences in biosynthetic rates and not breakdown rates. The
levels of several of the enzymes involved in the biosynthesis
were also different, though often not proportional or even
related to levels of biosynthesis.
When loganin (an intermediate of the terpenoid indole alkaloid
biosynthetic pathway) was added to the cultures, alkaloid
biosynthesis was increased in both cell lines (from 5 to 100
times). The levels of all the biosynthetic enzymes investigated
remained the same. From these results I concluded that the
amounts of the enzymes I measured did not limit terpenoid indole
alkaloid accumulation under normal culture conditions, since the
same levels could give rise to higher fluxes when precursor was
added. In mca terminology: their flux control coefficient is low
(but I can not really calculate them can I?). Another conclusion
was that enzymes with higher flux control coefficients will be
found proximal (before) the intermediate loganin.
Since I do not work directly with gene technology and if I would
start to, I still know it is quite laborious, I am trying to find
a way to avoid it but still use mca. Is this possible?
Is it possible to consider an added intermediate of the pathway
under study a parameter and calculate its response coefficient
and so locate the enzymes which will have higher flux control
coefficients? I have not yet found any similar experiment using
mca.
Thank you in advance for all comments and suggestions.
Denise Dagnino
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Denise Dagnino
The Technical University of Denmark
Dept. of Organic Chemistry
Building 201
DK-2800 Lyngby
Denmark
tel: **-45-45252111
fax: **-45-45933968
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