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1 Οκτ 2013 (πριν από 3 χρόνια και 6 μήνες)

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DEPARTMENT OF

COMPUTATIONAL MEDICI
NE

&

BIOINFORMATICS

SEMINAR


Wednesday
December 5
, 201
2

3:
3
0 PM


4:
3
0 PM

Forum Hall
, 4
th

floor, Palmer Commons



Robust Metabolic Homeostasis in Reconstituted Biochemical Reaction
Networks



Presented by:


Dr.
Alex Ninfa

Professor, Biological Chemistry

Department of Biological Chemistry

University of Michigan Medical School


Escherichia coli

and related bacteria maintain balanced metabolism in different environments, in part due to robust m
etabolic
homeostasis of the ratio of glutamine to a
-
ketoglutarate. When ammonium is available to cells, this metabolic homeostasis is
due in part to the UTase/UR
-
PII
-
ATase
-
GS cascade that controls the activity of glutamine synthetase (GS) by reversible
ad
enylylation. Here, we used a purified glutaminase that converts glutamine to glutamate and ammonia to simulate cellular
demand for glutamine. When purified GS was balanced against glutaminase in a reconstituted system containing excess
substrates (ATP, a
mmonium, glutamate), the reconstituted regulatory cascade provided approximate homeostasis of the
glutamine concentration by changing the adenylylation state of GS to compensate for changes in the total concentration of
GS or glutaminase, or the presence o
f a GS inhibitor. When the level of glutaminase and other conditions were constant, the
reconsituted system maintained a constant concentration of unmodified GS subunits, regardless of the total GS concentration.

This shows how
E. coli

balances GS activi
ty against the demand for glutamine in nitrogen
-
rich conditions.


Using the reconstituted biochemical networks, we explored the robustness of homeostasis to parameter variation. These
studies indicated a parameter responsible for setting the glutamine lev
el of the system, and suggested that a high kinetic
order of the steady state glutamine response of the regulatory system was correlated with the ability to maintain homeostasis

of the glutamine concentration.


In contradiction of a paper from the Alon gro
up, we show that avidity in the binding of ATase to GS had no role in the ability of
the reconstituted systems to maintain a constant concentration of unmodified GS subunits. We eliminated the possibility of
avidity by replacing the wild
-
type bifunctional

ATase enzyme in the reconstituted system with a pair of complementary
monofunctional enzymes, each containing only a single active site able to bind to GS. Using the monofunctional enzymes in
reconstituted networks, we observed normal homeostasis of unmo
dified GS subunits and glutamine concentration as the total
GS concentration was varied, counterindicating a requirement for avidity in the ATase binding to GS for robust homeostasis.


In the course of our studies, we also observed that the sensitivity of
purified GS to feedback inhibition was not altered by
reversible adenylylation, as widely believed. Instead, we observed that under the conditions of our experiments,
adenylylation of GS subunits completely inactivated the modified subunit without any maj
or effects on the feedback inhibition
properties of the other subunits in the GS dodecamer.


Together, our results show that the reconstituted UTase/UR
-
PII
-
ATase
-
GS cascade system has the ability to maintain
approximate homeostasis of the concentration of
unmodified GS subunits and glutamine, and this ability does not depend on
avidity in the binding of ATase to GS or on synergistic effects of GS adenylylation and feedback inhibition. However, the
reconstituted system did not display the ability to maintai
n a constant ratio of glutamine to a
-
ketoglutarate as the latter was
varied; instead only modest changes in the glutamine concentration occurred as a
-
ketoglutarate was varied throughout its
physiological range. This suggests that the maintainence of a con
stant ratio of glutamine to a
-
ketoglutarate is an emergent
property of the cell, with contributions from multiple systems.