Tài liệu Báo cáo khoa học: Quantitative estimation of channeling from early glycolytic intermediates to CO2 in intact Escherichia coli pdf

Quantitative estimation of channeling from early glycolytic
intermediates to CO
2
in intact Escherichia coli
Georgia Shearer, Jennifer C. Lee, Jia-an Koo and Daniel H. Kohl
Department of Biology, Washington University, St. Louis, MO, USA
The idea that intermediates in many metabolic path-
ways are ‘channeled’ from one pathway enzyme to the
next is widely [1,2], but not universally, accepted. One
reason for the controversy is that ‘many of the enzyme
complexes are dissociated during isolation owing to
dilution effects’ [3]. Srere, in his authoritative 1987
review [4], critically examined the evidence to that

date. For more recent reviews, see [1,5]. Contrarians,
such as Gutfreund and Chock [6], interpret their
kinetic data, compiled from experiments with pure
enzymes of the glycolytic pathway in dilute solution,
to be compatible with a diffusion model without need
to invoke channeling.
Atkinson [7] was influential in preparing the ground
for the idea of channeling. He pointed out that there is
not enough water in the cell to support uniform con-
centrations of all pathway intermediates at K
M
, the

approximate concentration traditionally assumed to be
necessary to permit pathways to function optimally.
Along with other considerations, this led Srere to
postulate the existence of ‘metabolons’, transient asso-
ciations of pathway enzymes in addition to stable
complexes (e.g. cytochrome complexes of the electron
transport chain or the covalent linkage of tryptophan
synthase subunits). In a metabolon, the presumption is
that the proximity of sequential enzymes would cause
Keywords
glycolysis; metabolic channelling;
metabolon; ratio of channeled flux to total

flux
Correspondence
D. H. Kohl, Department Biology,
Washington University, St. Louis,
MO 63130, USA
Fax: +1 314 935 4432
Tel: +1 314 935 5387
E-mail:
Website: />(Received 30 December 2004, revised 31
March 2005, accepted 7 April 2005)
doi:10.1111/j.1742-4658.2005.04712.x
A pathway intermediate is said to be ‘channeled’ when an intermediate just

made in a pathway has a higher probability of being a substrate for the
next pathway enzyme compared with a molecule of the same species from
the aqueous cytoplasm. Channeling is an important phenomenon because
it might play a significant role in the regulation of metabolism. Whereas
the usual mechanism proposed for channeling is the (often) transient inter-
action of sequential pathway enzymes, many of the supporting data come
from results with pure enzymes and dilute cell extracts. Even when isotope
dilution techniques have utilized whole-cell systems, most often only a
qualitative assessment of channeling has been reported. Here we develop a
method for making a quantitative calculation of the fraction channeled in
glycolysis from in vivo isotope dilution experiments. We show that fruc-
tose-1,6-bisphosphate, in whole cells of Escherichia coli, was strongly chan-

neled all the way to CO
2
, whereas fructose-6-phosphate was not. Because
the signature of channeling is lost if any downstream intermediate prior to
CO
2
equilibrates with molecules in the aqueous cytosol, it was not possible
to evaluate whether glucose-6-phosphate was channeled in its transforma-
tion to fructose-6-phosphate. The data also suggest that, in addition to
pathway enzymes being associated with one another, some are free in the
aqueous cytosol. How sensitive the degree of channeling is to growth or
experimental conditions remains to be determined.

Abbreviations
Fru1,6P
2
, fructose-1,6-bis phosphate; Fru6P, fructose-6-phosphate; F
ch
, fraction of total flux that is channelled; Glu6P, glucose-6-
phosphate; OPPP, oxidative limb of the pentose phosphate pathway; PFK, phosphofructokinase; PGI, phosphoglucoisomerase;
TCA, tricarboxyclic acid.
3260 FEBS Journal 272 (2005) 3260–3269 ª 2005 FEBS
the product of the first enzyme to have an advantage
in competition for the active site of the second enzyme
compared with the same molecular species within the

aqueous cytoplasm of the cell. That is, intermediates
just made in a pathway are not part of the same pool
as are identical molecules within the cell. Intermediates
produced within the pathway are ‘channeled’ to the
next enzyme.
Evidence for channeling
Prior investigations of channeling may be divided into
two categories: in vitro and in vivo. The former include
experiments with cell extracts and purified enzymes.
These are inherently unsatisfying. In particular, chan-
neling is thought to be the result of protein–protein
interactions in the crowded, organized environment of

the cell. Although investigators sometimes attempt to
simulate the crowding within a cell by, for example,
adding polyethylene glycol, the complexity and organ-
ization of the system is lost. Masters [8] emphasized
the point by noting that ‘many of the conditions com-
monly employed … are biologically abnormal and bear
little relation to the conditions under which enzymes
must act in vivo’. Among tools used for investigating
channeling are the following.
In vitro experiments
(a) Chromatographic techniques. Beeckmans [9]
reviewed in great detail the results of experiments util-

izing chromatographic techniques. (b) Copurification
of sequential pathway enzymes; for example, Law and
Plaxton [10]. (c) Coprecipitation of sequential pathway
enzymes; for example, Datta et al. [11]. (d) Isotope
dilution; for example, Debnam et al. [12].
In vivo experiments
(a) Genetic manipulation to disrupt enzyme complexes.
Several reports from Srere’s laboratory [3] provide
absolutely convincing proof that the interaction of
malate dehydrogenase and citrate synthase are essential
for the functioning of the tricarboxylic acid (TCA)
cycle at its usual rate. (b) Electron microscopy. Micro-

graphs showing colocalization of enzymes of the urea
cycle across the mitochondrial membrane [13] are con-
sistent with the proximity of sequential enzymes that is
evoked as a necessary (although not sufficient) condi-
tion for channeling. (c) NMR. Incubation of yeast in
[4-
13
C]glutamate did not result in the randomization of
the label in aspartate formed from it as would be
expected if the symmetric intermediates, succinate
and ⁄ or fumarate, dissociated from their enzymes and
were free to rotate [14]. In addition

19
F NMR studies
of citrate synthase 1 tagged with 5-fluorotryptophan
showed motional restriction in vivo [15]. (d) The use of
stable isotopes. Clegg and Jackson [16] compared the
specific activity of
14
C-labelled glycolytic intermediates
with that of pyruvate. These studies resulted in much
less dilution of the radioactivity in pyruvate than
would be expected if intermediates dissociated from
their enzymes and entered the cytosol. (In order to

facilitate uptake of intermediates these investigators
permeabilized the cells with dextran sulfate.)
The body of work cited above is strong evidence of
channeling in a number of pathways; glycolysis, the
TCA cycle, the oxidative limb of the pentose phos-
phate pathway (OPPP), the urea cycle. However, these
investigations do not provide data that can be used to
calculate the fraction of the flux through a pathway
that is channeled. The experimental designs used by
Negrutskii and Deutscher [17] result in data that could
be used to calculate the quantitative importance of
channeling in a specific pathway. They used Chi-

nese hamster ovary cells in studies of channeling of
aminoacyl-tRNA for protein synthesis. The cells were
electroporated to facilitate entry of
3
H-labeled amino-
acyl-tRNA and
14
C-labeled free amino acids. The quan-
tities of [
14
C]aminoacyl-tRNA (made in the pathway
from

14
C-labeled amino acids), [
3
H]aminoacyl-tRNA,
14
C- and
3
H-labeled protein were measured. The
3
Hin
protein was insignificant, showing that the aminoacyl-
tRNA made from amino acids and tRNA did not mix

with the introduced aminoacyl-tRNA; i.e. there was
perfect channeling from free amino acids to protein. In
this experiment, it was not necessary to calculate the
percentage of the flux that was channeled, because the
unchanneled flux was essentially zero. However, had
channeling been less than 100%, the data collected
would have enabled this calculation.
In this study, we describe experiments aimed at cal-
culating the fraction of the flux from early glycolytic
intermediates to CO
2
in intact Escherichia coli. Cells

incubated with [
14
C]glucose made
14
C-labeled glyco-
lytic intermediates. When the incubation mix also
included a [
12
C]intermediate, assuming that this inter-
mediate entered the cell, there was a competition
between the intermediate just produced in the pathway
and the same molecular species in the aqueous cytosol.

To the degree the latter was successful, the amount of
14
CO
2
evolved was decreased. Cells were also incuba-
ted in [
12
C]glucose plus [
14
C]intermediate, [
14
C]glucose

alone and [
14
C]intermediate alone. Combining these
data allowed us to calculate the fraction of the total
flux to CO
2
that was channeled. We used an E. coli
G. Shearer et al. Quantitation of channeling in intact E. coli
FEBS Journal 272 (2005) 3260–3269 ª 2005 FEBS 3261
mutant that was engineered to take up 6-carbon sugar
phosphates constitutively. The results indicate a high
degree of channeling of fructose 1,6-bisphosphate

(Fru1,6P
2
)toCO
2
(99 ± 16% of the flux from
Fru1,6P
2
channeled, P ¼ 0.005). This result requires
that all of the downstream steps from Fru1,6P
2
to
pyruvate and the further oxidation of pyruvate to CO

2
(presumably via mixed acid fermentation) be 100%
channeled. We found no significant channeling of
Glu6P or Fru6P to CO
2
. However, because Fru6P
was not channeled, the signature of any channeling of
Glu6P that might exist would be lost.
Results
The cells grew well on all carbon sources of interest to
us, including the 6-carbon sugar phosphates, Glu6P,
Fru6P, and Fru1,6P

2
. The doubling times for Glu6P
and Fru6P were comparable with the doubling time
of the parental strain growing on glucose (Table 1).
Doubling times when grown on glycerol and Fru1,6P
2
were considerably longer.
Rate of
14
CO
2
evolution and growth rate were

poorly correlated. In particular, the rate of
14
CO
2
evo-
lution from Fru1,6P
2
was twice that for Glu6P and
Fru6P, whereas the doubling time of cells grown on
the former was about twice as long as it was for
growth on the latter two.
The evolution of

14
CO
2
from [U-
14
C]glucose by the
mutant strain began with no lag and was linear with
time when cell density in the incubation mix was
A
600
¼ 0.8. Thus, at this density the pathways produ-
cing

14
CO
2
were in steady state. Above that density,
the rate of
14
CO
2
evolution increased with time (data
not shown).
The amounts of
14

CO
2
evolved in all incubations
were highly correlated with the total amounts of
14
C
taken up into the cell (three experiments, four treat-
ments in each experiment, three or four replicates each
treatment ¼ 40 data points; R
2
¼ 0.97, data not
shown). The total amount of

14
C entering the cell was
taken to be the sum of the
14
CO
2
evolved plus the
amount retained by the cell.
Saturation of
14
CO
2

evolution rate by the mutant
strain as a function of concentration depended on sub-
strate. The rate of
14
CO
2
evolution was saturated when
the concentration of glucose labeled with
14
C was 1 mm,
although it was not saturated until the concentration
reached 5 mm [

14
C]Glu6P or 10 mm [
14
C]Fru6P or
[
14
C]Fru1,6P
2
(data not shown).
Experiments to determine pathway allocation
of CO
2

evolution
Using the data shown in Table 2 and calculating as
described in Experimental procedures, about 96% of
the CO
2
was evolved via oxidation of pyruvate fol-
lowed by mixed acid fermentation and the TCA cycle.
Therefore, we have ignored the CO
2
evolved by the
OPPP when making calculations of the fractional
importance of channeled flux in glycolysis.

Experiments to investigate channeling of early
glycolytic intermediates to CO
2
Figure 1 illustrates our experimental paradigm. In this
example, the cells are incubated in [
14
C]glucose and
[
12
C]-Fru6P (the challenger). This creates a competi-
tion for E3 (phosphofructokinase; PFK, EC 2.7.1.10).
The measure of channeling is the degree to which the

Fru6P just made in the pathway is disproportionately
successful in being the substrate for PFK. Invoking the
usually proposed mechanism for channeling, the pref-
erence for the intermediate just made in the pathway is
a consequence of the interaction of PFK and the prior
enzyme in the pathway, phosphoglucoisomerase (PGI,
EC 5.3.1.9). If all Fru6P molecules just made in the
pathway dissociate from PGI and equilibrate with the
pool of Fru6P in the aqueous cytoplasm, then there
would be no channeling. When there is no channeling,
the result of the competition for binding to PFK will
be proportional to the number of [

14
C]Fru6P mole-
cules made in the pathway and the [
12
C]Fru6P in the
aqueous cytoplasm. In principle, the challenger can be
any glycolytic intermediate or any compound that, on
entry into the cell, is converted to a glycolytic inter-
mediate (e.g. mannose to mannose-6-phosphate to
Fru6P). In addition, results from the inverse experi-
ment ([
12

C]glucose vs. [
14
C]challenger) are necessary
for calculating the fraction of the total flux that is
channeled, as discussed later. In this regard, note that,
in addition to the putative interaction of two or more
Table 1. Growth of E. coli and
14
CO
2
evolution during incubation
with [U-

14
C]carbon source, each at 1 mM.
C source Strain
Doubling
time (h)
14
CO
2
evolution
during 30 min
incubation (37 °C)
(nmolÆmin

)1
)
Glucose RK 9118 parent 2.62
Glucose RK9117 mutant 2.23 ± 0.32 0.283 ± 0.014
Glycerol RK9117 mutant 3.61 ± 0.42
Glu6P RK9117 mutant 1.77 0.053 ± 0.004
Fru6P RK9117 mutant 2.17 0.042 ± 0.001
Fru1,6P
2
RK9117 mutant 4.05 ± 0.72 0.092 ± 0.001
Quantitation of channeling in intact E. coli G. Shearer et al.
3262 FEBS Journal 272 (2005) 3260–3269 ª 2005 FEBS

enzymes, there are free enzymes whose activity would
result in unchanneled flux even if every intermediate
just made in the pathway remained within the putative
complex. Finally, if channeling is being assessed by
measuring the isotopic composition of a downstream
compound (such as CO
2
), the signature of channeling
will be lost if any step between the reaction being con-
sidered and the downstream intermediate is not chan-
neled. Thus, an experiment with our design will result
in an evaluation of the minimum channeling in any

intervening step.
Figure 2 shows the results of experiments in which
cells were incubated with [U-
14
C]glucose alone (black
bars) and with [U-
14
C]glucose plus unlabeled inter-
mediate (white bars).
Challenging the glycolytic product of [U-
14
C]glucose

with [U-
12
C]Fru1,6P
2
had no effect on the quantity
of
14
CO
2
evolved compared with incubation in
[U-
14

C]glucose alone (8.8 ± 1.8 vs. 7.9 ± 1.2 nmols).
Challenging the [
14
C]Glu6P made in the pathway from
[
14
C]glucose with [
12
C]Glu6P taken up by the cell
from the incubation mix resulted in only a modest, but
significant (P ¼ 0.044), decrease in the counts in CO
2

(8.2 ± 1.0 vs. 5.3 ± 0.6). By contrast, exogenous
[
12
C]Fru6P had a clear impact on the counts in CO
2
(21.2 ± 2.2 vs. 7.9 ± 0.8) originating from [
14
C]glu-
cose. This suggests that Fru1,6P
2
is strongly channeled
all the way to CO

2
, that Glu6P is modestly channeled
and that Fru6P is channeled to an even lesser degree.
But two additional, essential pieces of information are
required before even such a qualitative conclusion con-
cerning the degree of channeling can be drawn. Also
these data by themselves do not allow us to calculate
the fraction of the flux that is channeled. The first
additional requirement is to show that the exogenous
intermediate from the incubation mix entered the cell.
Clearly, if the exogenous intermediate did not enter
the cell, it could not compete for the active site of the

enzyme for which it is substrate. Data relevant to this
are shown in Fig. 3. These data establish that all of
the exogenous intermediates entered the cells (black
bars) and evolved CO
2
, although with varying degrees
of success. Cells grown in 2% glycerol evolved
14
CO
2
from Fru1,6P
2

at an even higher rate (32.4 ±
7.7 nmol per 30 min; Fig. 3, solid bar) than did the
same cells from glucose (8.8 ± 1.8 nmol per 30 min;
Fig. 2, solid bar). In the presence of glucose, cells
grown in 2% glycerol evolved almost no CO
2
from
Fru6P (data not shown). However when 25 lm Fru6P
was added to the growth medium, CO
2
was evolved
from Fru6P at a good rate (Fig. 3, solid bar). The

implied increase in uptake of Fru6P was unexpected
because the cells were engineered to be constitutive for
the uptake of 6-carbon sugar phosphates.
In addition to establishing the degree to which the
exogoenous intermediates enter the cell, the data of
Fig. 3 also show the consequence for
14
CO
2
evolution
of coincubating [
14

C]intermediates with [
12
C]glucose.
Whereas the [
12
C]glucose tended to decrease
14
CO
2
when [
12
C]glucose was coincubated with a [

14
C]inter-
mediate, the radioactivity in the CO
2
evolved was
significant compared to that in the absence of
[
12
C]glucose; 42, 72, and 67% for [
14
C]Glu6P,
[

14
C]Fru6P and [
14
C]Fru1,6P
2
, respectively. This is
not in conflict with the result that unlabeled Fru1,6P
2
did not dilute the radioactivity in
14
CO
2

when it was
coincubated with [
14
C]glucose (white bar vs. black
bar, Fig. 2). Despite the fact that exogenous Fru1,6P
2
entered the cell, it was unsuccessful in competing for
the catalytic site of aldolase (EC 4.1.2.13) with the
Fru1,6P
2
just made in the pathway originating from
[

14
C]glucose (Fig. 2, members of the third pair are
not significantly different). The data in Fig. 3 suggest
that some PFK molecules are associated with aldolase
and downstream enzymes in a metabolon, while, at
the same time, other molecules of aldolase and down-
stream glycolytic enzymes are free in the aqueous
cytoplasm. These free enzymes are able to convert the
exogenous [
14
C]Fru1,6P
2

to
14
CO
2
in the presence of
[
12
C]glucose.
The data in Figs 2 and 3, considered separately,
are insufficient to determine the predicted result if
Table 2. Fraction of the total flux to
14

CO
2
via the OPPP. Note that only one of the six carbon atoms in [1-
14
C]glucose and [6-
14
C]glucose
are labeled, whereas the label in [U-
14
C]glucose is divided among all six atoms. Glc, glucose; % of total flux to
14
CO

2
via the OPPP ¼
100 · 3.42 ⁄ 79.5 ¼ 4.3%.
[1-
14
C]Glucose [6-
14
C]Glucose
[1-
14
C]Glucose minus
[6-

14
C]glucose (OPPP) [U-
14
C]Glucose
[
14
C]Glucose converted to
14
CO
2
(%) 2.32 0.95 1.37 5.3
Nano equivalents

14
C per nmols [
14
C]glucose 1 1 1 6
nmol glucose initially present 250 250 250 250
Nano equivalents
14
C initially present 250 250 250 1500
nmol
14
CO
2

produced 5.80 2.38 3.42 79.5
G. Shearer et al. Quantitation of channeling in intact E. coli
FEBS Journal 272 (2005) 3260–3269 ª 2005 FEBS 3263
channeling were zero. When the data from both figures
are combined, we are able to calculate the expectation
if the fraction channeled were zero (F
ch
¼ 0). When
F
ch
¼ 0, the relative success of binding to the enzyme
of intermediates just made in the pathway and those in

the aqueous cytoplasm will be proportional to their
relative numbers. A method for calculating the fraction
of the flux that is channeled from each intermediate to
CO
2
, using the data of Figs 2 and 3, is developed
below.
Discussion
The first task is to calculate the relative amounts of
intermediate made in the pathway vs. the same inter-
mediate made from exogenous sources. In this regard,
Fig. 2. Effect of unlabeled challenging compound (Glu6P, Fru6P or

Fru1,6P
2
) on the quantity of
14
CO
2
evolved by E. coli cells when
incubated with [
14
C]glucose. Black bars represent the quantity
evolved when cells were incubated with [
14

C]glucose alone. White
bars represent the quantity evolved when cells were incubated
with [
14
C]glucose and unlabeled challenging intermediate (Glu6P,
Fru6P, or Fru1,6P
2
). For experiments with Fru6P, cells were grown
with a trace of Fru6P (25 l
M) in addition to 0.2% (v ⁄ v) glycerol as
the carbon source.
Fig. 3. Effect of unlabeled glucose on the quantity of

14
CO
2
evolved by E. coli cells when incubated with
14
C-labeled intermedi-
ates (Glu6P, Fru6P or Fru1,6P
2
). Black bars represent the quantity
evolved when cells were incubated with
14
C-labeled intermediate

alone; white bars represent the quantity evolved when cells were
incubated with
14
C-labeled intermediate and unlabeled challenging
glucose. For experiments with Fru6P, cells were grown with a
trace of Fru6P (25 l
M) in addition to 0.2% (v ⁄ v) glycerol as the car-
bon source.
Fig. 1. Glycolytic pathway in an E. coli cell coincubated with
[
14
C]glucose and a [

12
C]intermediate (Fru6P in this example).
[
14
C]Glucose is converted to [
14
C]Glu6P as it enters the cell via the
phosphotransferase system (PTS) and thence to [
14
C]Fru6P by E2
(phosphoglucoisomerase; PGI). In the absence of channeling,
[

14
C]Fru6P will equilibrate with [
12
C]Fru6P from the exogenous
source, competing with [
14
C]Fru6P for the catalytic site of E3
(phosphofructokinase; PFK), thereby decreasing the amount of
14
CO
2
that would have been evolved had no [

12
C]Fru6P had been
present. To the degree that the
14
C and
12
C intermediates do not
equilibrate, the amount of
14
CO
2
evolved will be decreased to a les-

ser degree, unless downstream
14
Cand
12
C intermediates equili-
brate.
Quantitation of channeling in intact E. coli G. Shearer et al.
3264 FEBS Journal 272 (2005) 3260–3269 ª 2005 FEBS
note that the amount of intermediate present at any
given time is proportional to the total uptake because
the system is in steady state as indicated by the con-
stant rate of the evolution of

14
CO
2
from the earliest
time point (see Results). Also recall from the Results
that the CO
2
evolved from [
14
C]glucose was strictly
proportional to the total uptake of glucose. Thus, we
can use CO

2
evolved as a reliable proxy for the total
uptake.
To facilitate the following discussion, let A ¼ the
14
CO
2
evolved when cells were incubated with [
14
C]glu-
cose alone; B ¼ the
14

CO
2
evolved when cells were
incubated with [
14
C]glucose plus [
12
C]exogenous inter-
mediate; C ¼ the
14
CO
2

evolved when cells were incu-
bated with [
14
C]exogenous intermediate alone; D ¼ the
14
CO
2
evolved when cells were incubated with
[
14
C]exogenous intermediate plus [
12

C]glucose.
The total amount of intermediate is the amount, for
example, of Fru6P that has just been made in the
pathway plus the exogenous Fru6P that entered the
cell. The amount of [
14
C]Fru6P converted to
14
CO
2
from [
14

C]glucose as the result of coincubation of
[
14
C]glucose and [
12
C]intermediate is proportional to
‘B’ as defined above. Likewise the amount of
[
14
C]Fru6P converted to
14
CO

2
from exogenous
[
14
C]Fru6P when coincubated with [
14
C]Fru6P is pro-
portional to ‘D’.
Consequently in the absence of channeling (i.e. when
the fraction of the flux that is channeled is zero,
F
ch

¼ 0), the expected dilution of
14
CO
2
originating
from
14
C-labeled glucose when challenged by unlabeled
intermediate, is equal to B ⁄ (B + D). That is, if
B ⁄ A ¼ B ⁄ (B + D), then F
ch
¼ 0.

When the exogenous intermediate is completely
unsuccessful in decreasing the radioactivity of the CO
2
evolved from [
14
C]glucose, then
B=A ¼ 1; and F
ch
¼ 1
In principle, the exogenous intermediate (say Fru6P)
could be channeled as the result, for instance, of its
permease interacting with PFK. By analogy with the

above if D ⁄ C ¼ D ⁄ (B + D), then F
ch
¼ 0.
Likewise, when the intermediate made from glucose
does not dilute the radioactivity of CO
2
from a coincu-
bation of [
12
C]glucose and [
14
C]intermediate, then

D=C ¼ 1; and F
ch
¼ 1
Values of F
ch
between 0 and 1 can be calculated by
interpolation as illustrated in Fig. 4. An important
caveat, if one is to make this interpolation, is that a
substantial quantity of the exogenous intermediate
must enter the cell during coincubation with glucose.
Clearly, the exogenous intermediate cannot compete
with the intermediate just made in the pathway for

occupancy at the active site of the appropriate enzyme
if it does not get into the cell. In our notation, if not
much of the intermediate gets into the cell when glu-
cose is present, then B » D and B ⁄ (B + D)  1. If the
expected value when there is no channeling is close to
1 and the value for 100% channeling is also 1, then
there is no ‘room’ to interpolate as required by the
procedure outlined in Fig. 4.
The data in Figs 2 and 3 permit calculation of
B ⁄ (B + D), the ratio by which
14
CO

2
evolved from
[
14
C]glucose would be affected by coincubation with
an unlabeled intermediate, if no channeling occurred,
compared with B⁄ A, the observed effect (Fig. 5).
When the exogenous intermediate was Fru1,6P
2
, there
was a large, significant difference between the observed
impact on

14
CO
2
evolution from [
14
C]glucose when
coincubated with [
12
C]Fru1,6P
2
(B ⁄ A) vs. the values
that would be expected if there were no channeling

Fig. 4. Scheme for calculating the fraction of the total flux that is
channeled (F
ch
).
Fig. 5. Expected fractional effect on
14
CO
2
evolved of coincubating
E. coli cells with [
14
C]glucose and unlabeled intermediate in the

absence of channeling [B ⁄ (B + D)] compared to the observed
effect (B ⁄ A). Black bars; the expected effect ¼ B ⁄ (B + D). White
bars; the observed effect ¼ B ⁄ A.
G. Shearer et al. Quantitation of channeling in intact E. coli
FEBS Journal 272 (2005) 3260–3269 ª 2005 FEBS 3265
[B ⁄ (B + D)] (Fig. 5). This result is consistent with the
Fru1,6P
2
just made in the pathway being channeled.
By contrast, for Glu6 P and Fru6P, Fig. 5 shows no
significant difference between the observed effect
(Fig. 5, Glu6P or Fru6P, white bar) and the value

expected if there were no channeling (Fig. 5, Glu6P or
Fru6P, black bar). This is a necessary but not suffi-
cient indication of the absence of channeling. The data
in Fig. 3 rule out the exogenous intermediate not
entering the cell as an explanation. There is a second
alternative explanation for the apparent absence of
channeling of Glu6P. Glu6P just made in the pathway
could be strongly channeled to Fru6P, but the signa-
ture of that channeling would be lost because Fru6P
was apparently not channeled.
The data in Table 3 show the method for and the
results of calculating the fraction of the total flux that

is channeled.
The most striking result is that essentially all of the
flux from Fru1,6P
2
to CO
2
was channeled (Table 3;
F
ch
¼ 0.99 ± 0.16). Note that this required almost
perfect channeling of each intermediate in the pathway
to pyruvate and in the reactions that oxidize pyruvate

to CO
2
. This is a surprising result.
When the paradigm of Fig. 4 is used to calculate F
ch
for Glu6P or Fru6P just made in the pathway, the
results are F
ch
¼ 0.10 ± 0.26 and 0.04 ± 0.01,
respectively. That is, the data are consistent with no
channeling. However, as noted above, Glu6P could be
strongly channeled to Fru6P but the signature would

be lost because Fru6P is not channeled. It would be
surprising if Glu6P were very strongly channeled in
glycolysis because it has other metabolic fates. In addi-
tion, Clegg and Jackson’s data [13] are consistent with
Glu6P, if it is channeled at all, being channeled to a
lesser degree than were the other intermediates tested.
It is theoretically possible that the appropriate
enzyme might ‘prefer’ the exogenous substrate (after it
enters the cell) to the intermediate just made in the
pathway. In the context of the usual model, this would
require interaction between the permease responsible
for the import of the exogenous intermediate and the

pathway enzyme responsible for its entry into glycoly-
sis. However, the data [D ⁄ (B + D) vs. D⁄ C] provide
no evidence for channeling from any exogenous Glu6P
or Fru1,6P
2
to CO
2
. Although there was a significant
difference between D ⁄ (B + D) vs. D ⁄ C for Fru6P, the
fraction channeled was small (0.16 ± 0.01).
The evidence presented here for channeling of
Fru1,6P

2
to CO
2
is consistent with the existence of a
glycolytic complex that holds together long enough for
the Fru1,6P
2
that binds to it to be converted through
Table 3. Calculation of the fraction of the total flux that is channeled (F
ch
). Values are means ± SE.
Challenging intermediate Glu6P Fru6P Fru1,6P

2
No. experiments 6 2 5
Carbon source in growth medium 22 m
M glycerol 22 mM glycerol
+25 l
M Fru6P
22 m
M glycerol
A (nmol
14
CO
2

evolved from incubation
with [
14
C]glucose alone)
8.2 ± 1.0 21.2 ± 2.2 8.8 ± 1.8
B (nmol
14
CO
2
evolved from incubation
with [
14

C]glucose plus unlabeled intermediate)
5.3 ± 0.6 7.9 ± 0.8 7.9 ± 1.2
C (nmol
14
CO
2
evolved from incubation
with [
14
C]intermediate alone)
6.4 ± 1.7 22.0 ± 1.8 32.4 ± 7.7
D (nmol

14
CO
2
evolved from incubation
with
[14
C]intermediate plus unlabeled glucose)
2.7 ± 1.0 15.8 ± 2.6 21.6 ± 5.9
Evaluation of channeling of intermediate just made in the pathway
B ⁄ (B + D) (expected effect of unlabeled
challenger on
14

CO
2
evolution from
[
14
C]glucose if no channelling
0.695 ± 0.090 0.333 ± 0.004 0.311 ± 0.050
B ⁄ A (observed effect) 0.707 ± 0.119 0.358 ± 0.001 0.961 ± 0.104
F
ch
0.103 ± 0.256 (NS) 0.036 ± 0.012 (NS) 0.994 ± 0.158
(P ¼ 0.005)

Evaluation of channeling of exogenous intermediate
D ⁄ (B + D) (expected effect of unlabeled
challenger on
14
CO
2
evolution from
14
C-labeled intermediate if no channelling
0.305 ± 0.090 0.667 ± 0.008 0.689 ± 0.053
D ⁄ C (observed effect 0.386 ± 0.051 0.719 ± 0.005 0.637 ± 0.047
F

ch
0.065 ± 0.089 (NS) 0.155 ± 0.006 (P ¼ 0.017) 0.229 ± 0.145 NS
Quantitation of channeling in intact E. coli G. Shearer et al.
3266 FEBS Journal 272 (2005) 3260–3269 ª 2005 FEBS
the subsequent steps of glycolysis and on to CO
2
. Such
a putative complex has elements in common with the
complex proposed by Mowbray and Moses [18] on the
basis of observations using E. coli extracts. Observa-
tions by Clegg and Jackson [16] support the conclusion
that channeling of several glycolytic intermediates in

pemeabilized mouse fibroblasts occurred, but that this
channeling was somewhat leaky. However, in contrast
to the results reported here for E. coli, Fru1,6P
2
was
not strongly channeled in mouse L-929 cells.
Our results show that, under certain conditions,
E. coli cells channel an intermediate (Fru1,6P
2
) from
early in the glycolytic pathway all the way to CO
2

. Whe-
ther channeling over such a large number of intermedi-
ates is sensitive to growth conditions or other details of
the experimental protocol remains to be determined.
Experimental procedures
Materials
[U-
14
C]-, [1-
14
C]- and [6-
14

C]glucose, [U-
14
C]Glu6P, [U-
14
C]
Fru6P, and [U-
14
C]Fru1,6P
2
, as well as all other biochemi-
cals, were obtained from Sigma (St. Louis, MO). Our colla-
borator, Robert Kadner (University of Virginia), made an

E. coli mutant (RK9117) that was engineered to take up
4-, 5- and 6-carbon sugar phosphates constitutively. Table 4
gives the genotype of RK9117 and the parent from which it
was made (RK9118).
Bacterial growth
Cells were grown at 37° in a rotary shaker in a defined
medium [10.5 g K
2
HPO
4
, 4.5 g KH
2

PO
4
, 1 g (NH
4
)
2
SO
4
,
0.5 g trisodium citrate, 10 mm MgSO
4
,1mm CaCl

2
,5mg
thiamine and 0.2% (v ⁄ v) glycerol in 1 L] to an absorbance
at 600 nm of 0.8. In cultures to be used for channeling
experiments with Fru6P, the growth medium was supple-
mented with 25 lm Fru6P. This substantially increased the
amount of Fru6P taken up by the cells in the presence of
glucose. Cells were harvested by centrifugation (10 000 g,
10 min, 8 °C) and washed with growth medium free of
carbon source and washed pellets were stored at )80° until
needed.
Incubation conditions

Just before each experiment, cells were resuspended in a vol-
ume of growth medium (lacking any carbon source or Mg
2+
or Ca
2+
) such that the absorbance was  10 at 600 nm.
About 20 l L of the concentrated cells were added to 230 lL
of incubation medium in order to incubate cells at an absorb-
ance at 600 nm of 0.8. Each incubation mixture contained
appropriate carbon sources, one labeled with
14
C and, when

required by the experimental design, a second unlabeled car-
bon source. Incubations were carried out in 25 mL vials that
were sealed with a rubber septum fitted with a straight pin. A
small strip of filter paper was placed on the pin and wetted
with 10 lL 10% (w ⁄ v) NaOH. Incubations were carried out
for 30 min at 37 °C in a rotary shaker water bath. Incuba-
Table 4. Genotypes of E. coli strains used in this report.
Strain Genotype
RK9117 D(argF-lac)U169 araD139 thi gyrA219 relA rpsL150 non polA1 D(ilvBN-uhpABCT ¢)2095 zig621::Tn10 uhpA
+
B
+

C91::4 (Con)T
+
Rk9118 D(argF-lac)U169 araD139 thi gyrA219 relA rpsL150 non polA1 D(ilvBN-uhpABCT ¢)2095 zig621::Tn10 uhpA
+
B
+
C91::8 (Neg)T
+
Table 5. Effect of NaOH and filter paper on CPM and DPM of [U-
14
C]glucose. Each vial contained 9000 DPM [U-
14

C]glucose in 100 lLH
2
O,
plus the indicated additions. The greatest difference (9302 vs. 9059) is not statistically significant (P ¼ 0.12).
Vial
Filter
paper
NaOH
10 lL
Filter paper
plus NaOH CPM DPM
Mean

CPM ± SE
Mean
DPM ± SE
1 No No No 8350 8911 8488 ± 74 9059 ± 79
2 No No No 8603 9182
3 No No No 8511 9084
4 Yes No No 8542 9116 8554 ± 23 9135 ± 22
5 Yes No No 8521 9110
6 Yes No No 8599 9178
7 No Yes No 8609 9196 8611 ± 21 9198 ± 27
8 No Yes No 8648 9246
9 No Yes No 8575 9152

10 No No Yes 8648 9232 8719 ± 88 9302 ± 94
11 No No Yes 8616 9186
12 No No Yes 8894 9488
G. Shearer et al. Quantitation of channeling in intact E. coli
FEBS Journal 272 (2005) 3260–3269 ª 2005 FEBS 3267
tions were terminated by adding 100 lL 70% (v ⁄ v) HClO
4
.
The outgassed CO
2
was captured on the base impregnated fil-
ter paper. After incubation, the filter paper was removed and

eluted with 100 lL water. Three milliliters of scintillation
cocktail (Ecolite, ICN Biomedicals, Irvine, CA) were added.
Aliquots of the incubation mixture were also counted in
order to calculate percent conversion of the added
14
C-
source. The rate of CO
2
production was calculated as
nmolsÆmin
)1
¼ nmol equivalents of

14
C source · percentage
conversion ⁄ (100 · min).
The term ‘nmol equivalents of
14
C-source’ is included in
order to take into account the fact that six molecules of
CO
2
were produced from one molecule of glucose, Glu6P,
Fru6P or Fru1,6P
2

via glycolysis followed by mixed acid
fermentation. By contrast, only one molecule of CO
2
was
produced via the OPPP from one molecule of glucose or
Glu6P.
Radioactive counting
Radioactivity was measured by a Wallac (Model 1410)
liquid scintillation counter (PerkinElmer, Wellesley, MA,
USA) equipped with quench correction. Because 10 lLof
10% NaOH was present in samples used to measure CO
2

production, we did a control experiment to assess any
possible impact of quenching by NaOH. Ten microliters of
NaOH (10%) had no significant effect on either the counts
per minute (CPM) or the disintegrations per minute (DPM)
recorded by the counter (Table 5).
Estimation of the relative contribution of the
OPPP to the total CO
2
evolved
Because we were investigating channeling from early glyco-
lytic intermediates to pyruvate and on to CO
2

, any CO
2
produced via the oxidative limb of the OPPP must be cor-
rected for or shown to be small enough to neglect. Catabol-
ism of glucose via the OPPP results in CO
2
being evolved
from the C-1 position of glucose. When glucose is catabo-
lized completely to CO
2
in either the TCA cycle or by a
branch of mixed acid fermentation, then both the C-1 and

the C-6 positions of glucose give rise to CO
2
. Thus the
amount of CO
2
produced in the OPPP is CO
2
from
[1-
14
C]glucose minus CO
2

from [6-
14
C]glucose. The total
amount of CO
2
produced by the OPPP plus that produced
by the oxidation of pyruvate was calculated from incuba-
tions with [U-
14
C]glucose. The ratio of the above two
values is the fraction of CO
2

evolved by the OPPP.
Design of experiments to evaluate channelling
Incubations were carried out at 37 °C for 30 min as des-
cribed above. There were four treatments, each replicated
four times, in each experiment: (a) [U-
14
C]glucose (1 mm)
alone, (b) [U-
14
C]glucose (1 mm) plus
12
C challenging

intermediate (5 mm Glu6P or 5 mm Fru6P or 10 mm
Fru1,6P¼), (c) [U-
14
C]intermediate (5 mm Glu6P or 5 mm
Fru6P or 10 mm Fru1,6P
2
) alone, and (d) [U-
14
C]interme-
diate (5 mm Glu6P or 5 mm Fru6P or 10 mm Fru1,6P
2
)

plus [
12
C]glucose (1 mm).
Acknowledgements
This work was supported by an SGER NSF grant:
NSD Grant # MCB-02004900.
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