dbk at aber.ac.uk ("Douglas Kell") wrote in message news:<3BB4AAA7.8790.DF1D392A at localhost>...
> We are trying to get hold of delta-G (or K-eq) values for common
> biochemical reactions to see which are <ahem> 'irreversible'. There
> is a special interest in trp biosynthesis right now. Does anyone know
> where these can easily be got?
>> Many thanks,
> Douglas.
Hi,
Unfortunately I do not have a ready list of equilibrium constants. I
do wonder, though, how justified it would be to take {\Delta}G or
K_{eq} values obtained in the test tube and use them to understand
cellular processes. It seems that, in certain documented cases,
thermodynamic equilibria for component enzyme reactions are not useful
at all. The cellular processes seem to be under kinetic control, not
under thermodynamic control.
For example, consider the equilibrium constants for the binding of
zinc to various proteins. Zinc dissociation constants for zinc enzymes
typically range from only 10-6 to 10-12 M. Based on the total amount
of zinc in cells, these values of would suggest that there is a large
pool of free (unbound) zinc in the cytoplasm.
However, it is now known that the cells contain relatively large
amounts of zinc transport proteins with binding constant in the
femtomolar range (10-15 M). To quote from the article attached
below:
"[The] results contradict the long-held assumption that pools of free
zinc--and of other transition metals, such as copper--exist in cells.
[...] [Zinc]--and other transition metals--are always held tightly by
biopolymers and [...] their trafficking within cells must be
kinetically controlled by metalloregulatory proteins and perhaps by
other factors, such as metal chaperones. These are proteins that can
mobilize bound metal and conduct it to sites where it is required.
[...] It appears that zinc does not necessarily end up in the tightest
binding sites, as would be the case with simple thermodynamic
control."
Response, anyone?
- Petr
---------------------------------------------------
P e t r K u z m i c, Ph. D. * BioKin Ltd.
1652 S. Grand Ave. * Suite 337 * Pullman, WA 99163
509.334.4131 * 509.332.3493 fax * http://www.biokin.com
__________________________________________________
CHEMICAL & ENGINEERING NEWS
September 17, 2001
Volume 79, Number 38, p. 53
http://pubs.acs.org/email/cen/html/100701105549.html
NO POOLS OF FREE ZINC IN CELLS
Control of zinc trafficking in cells must involve metalloregulatory
proteins, recent studies suggest
MAUREEN ROUHI
--------------------------------------------
A bacterial cell contains up to 400,000 atoms of zinc. But when
graduate student Caryn E. Outten and chemistry professor Thomas V.
O'Halloran at Northwestern University searched for these atoms, they
found none floating freely in the cell cytosol.
Their results contradict the long-held assumption that pools of free
zinc--and of other transition metals, such as copper--exist in cells.
According to this idea, metal-requiring proteins coming off ribosomes
would pick and choose from these pools the metals they need.
"These ideas are now falling apart," O'Halloran says. Work in his lab
is leading him to believe that zinc--and other transition metals--are
always held tightly by biopolymers and that their trafficking within
cells must be kinetically controlled by metalloregulatory proteins and
perhaps by other factors, such as metal chaperones. These are
proteins that can mobilize bound metal and conduct it to sites where
it is required.
O'Halloran, in collaboration with Valeria C. Culotta, a professor of
environmental health sciences at Johns Hopkins University, had shown
before that copper chaperones help deliver copper to copper-requiring
proteins (C&EN, Nov. 10, 1997, page 34). Although these results
initially were met with skepticism, chaperoning copper seems necessary
to protect the cells from the metal's toxicity. But zinc is far less
toxic and far more abundant in cells than is copper. Chaperoning
based on toxicity seems unnecessary. Metal chaperones might be
involved "not because of the toxicity of metals, but because there are
a myriad of potential binding sites in cells," O'Halloran says.
To measure the amount of free zinc in cells, O'Halloran and Outten,
now a postdoctoral researcher in Culotta's lab, isolated two
metalloregulatory proteins that act as zinc sensors from Escherichia
coli and determined their responses to controlled concentrations of
zinc in vitro. One, called Zur, represses expression of a protein
involved in zinc uptake when the cell has just enough zinc. The
other, called ZntR, stimulates expression of a protein that removes
zinc when the cell has too much.
IN a test tube, Zur triggers the shutting down of zinc uptake when the
free zinc concentration is around 10-16 M, while ZntR triggers zinc
removal when free zinc is around 10-15 M [Science, 292, 2488 (2001)].
These concentrations in the maximum possible volume of a bacterial
cell, about 2 to 4 femtoliters, translate to less than one atom per
cell. "From a thermodynamic point of view, at any moment there's no
free zinc in the cytoplasm under normal growth conditions," O'Halloran
says. On average, the zinc must always be bound in sites throughout
the cell, he adds.
That's not surprising, given that the E. coli genome has at least 50
genes that encode zinc-binding proteins. Also, many cell
constituents, such as DNA, offer nonspecific binding sites of moderate
affinity.
In very recent work, Outten, O'Halloran, and postdoctoral research
associate Yutaka Hitomi have found that the dissociation constant of
zinc binding to ZntR is 10-15 M [J. Am. Chem. Soc., 123, 8614
(2001)]. That means ZntR binds zinc more avidly than other
zinc-binding proteins, such as carboxypeptidase and carbonic
anhydrase. Zinc dissociation constants for zinc enzymes typically
range from only 10-6 to 10-12 M.
To try to understand the basis of the tight zinc binding of these
sensors, the Northwestern team got help from chemistry professor James
E. Penner-Hahn, an expert in spectroscopic methods for determining
the coordination environment of metals in proteins, and graduate
student Daniel A. Tobin at the University of Michigan, Ann Arbor.
They found that Zur has two binding sites, one binding zinc more
tightly than the other. Sulfur and nitrogen or oxygen atoms make up
the coordination environment of zinc in these sites, with a higher
proportion of sulfur atoms in the tighter binding site [Biochemistry,
40, 10417 (2001)]. The roles of the two binding sites are not yet
clear, but O'Halloran notes that similar pairs of binding sites have
been found in other zinc sensor proteins.
Given the tight binding of zinc to ZntR, it's difficult to visualize
how zinc chemistry in the cytoplasm would operate under strict
thermodynamic control. In that scenario, ZntR will get the zinc atoms
first, because it has the tightest binding sites. As soon as it gets
saturated, it will trigger zinc disposal. But the concentration of
free zinc at which it triggers zinc disposal is at least three orders
of magnitude lower than that required to get zinc to other
zinc-requiring proteins. If those other proteins don't get the zinc
they need, the cell's work won't get done. A certain portion of the
pool of bound zinc has to be mobilized and inserted into new
zinc-requiring proteins as they are made.
"We don't know how the zinc gets into the correct sites," O'Halloran
says. "We suspect by analogy to the copper chaperone story that there
are factors that help facilitate the entry and removal of the metal at
the right time."
It appears that zinc does not necessarily end up in the tightest
binding sites, as would be the case with simple thermodynamic control.
Nor does zinc necessarily stick with the first binding site that it
encounters. "We suggest that metal chaperones have to be there to
mobilize zinc and conduct it to sites that it may not find in its
initial encounters but where the cell needs it," O'Halloran says.
__________________________________________________
