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

However, because Fru6P was not channeled, the signature of any channeling of Glu6P that might exist would be lost.. This suggests that Fru1,6P2 is strongly channeled all the way to CO2,

intermediates to CO2 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 KM, 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: kohl@biology.wustl.edu

Website: http://www.biology.wustl.edu/

(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 CO2, whereas fructose-6-phosphate was not Because the signature of channeling is lost if any downstream intermediate prior to

CO2 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 additransforma-tion 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.

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 Incubacondi-tion of yeast in

[4-13C]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 19F 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 14C-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 3H-labeled amino-acyl-tRNA and14C-labeled free amino acids The quan-tities of [14C]aminoacyl-tRNA (made in the pathway from 14C-labeled amino acids), [3H]aminoacyl-tRNA,

14C- and3H-labeled protein were measured The3H in protein was insignificant, showing that the aminoacyl-tRNA made from amino acids and aminoacyl-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 CO2 in intact Escherichia coli Cells incubated with [14C]glucose made 14C-labeled glyco-lytic intermediates When the incubation mix also included a [12C]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

14CO2 evolved was decreased Cells were also incuba-ted in [12C]glucose plus [14C]intermediate, [14C]glucose alone and [14C]intermediate alone Combining these data allowed us to calculate the fraction of the total flux to CO2 that was channeled We used an E coli

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

(Fru1,6P2) to CO2 (99 ± 16% of the flux from

Fru1,6P2 channeled, P¼ 0.005) This result requires

that all of the downstream steps from Fru1,6P2 to

pyruvate and the further oxidation of pyruvate to CO2

(presumably via mixed acid fermentation) be 100%

channeled We found no significant channeling of

Glu6P or Fru6P to CO2 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 Fru1,6P2 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 Fru1,6P2

were considerably longer

Rate of 14CO2 evolution and growth rate were

poorly correlated In particular, the rate of14CO2

evo-lution from Fru1,6P2 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 14CO2 from [U-14C]glucose by the

mutant strain began with no lag and was linear with

time when cell density in the incubation mix was

A600¼ 0.8 Thus, at this density the pathways

produ-cing 14CO2 were in steady state Above that density,

the rate of 14CO2 evolution increased with time (data

not shown)

The amounts of 14CO2 evolved in all incubations

were highly correlated with the total amounts of 14C

taken up into the cell (three experiments, four

treat-ments in each experiment, three or four replicates each

treatment¼ 40 data points; R2¼ 0.97, data not shown) The total amount of 14C entering the cell was taken to be the sum of the 14CO2 evolved plus the amount retained by the cell

Saturation of 14CO2 evolution rate by the mutant strain as a function of concentration depended on sub-strate The rate of 14CO2 evolution was saturated when the concentration of glucose labeled with14C was 1 mm, although it was not saturated until the concentration reached 5 mm [14C]Glu6P or 10 mm [14C]Fru6P or [14C]Fru1,6P2(data not shown)

Experiments to determine pathway allocation

of CO2evolution Using the data shown in Table 2 and calculating as described in Experimental procedures, about 96% of the CO2 was evolved via oxidation of pyruvate fol-lowed by mixed acid fermentation and the TCA cycle Therefore, we have ignored the CO2 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 CO2

Figure 1 illustrates our experimental paradigm In this example, the cells are incubated in [14C]glucose and [12C]-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 [14C]Fru6P mole-cules made in the pathway and the [12C]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 ([12C]glucose vs [14C]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 CO2evolution during incubation

with [U-14C]carbon source, each at 1 m M

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

Fru1,6P 2 RK9117 mutant 4.05 ± 0.72 0.092 ± 0.001

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 CO2), 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-14C]glucose alone (black

bars) and with [U-14C]glucose plus unlabeled

inter-mediate (white bars)

Challenging the glycolytic product of [U-14C]glucose

with [U-12C]Fru1,6P2 had no effect on the quantity

of 14CO2 evolved compared with incubation in

[U-14C]glucose alone (8.8 ± 1.8 vs 7.9 ± 1.2 nmols)

Challenging the [14C]Glu6P made in the pathway from

[14C]glucose with [12C]Glu6P taken up by the cell

from the incubation mix resulted in only a modest, but

significant (P¼ 0.044), decrease in the counts in CO2

(8.2 ± 1.0 vs 5.3 ± 0.6) By contrast, exogenous

[12C]Fru6P had a clear impact on the counts in CO2

(21.2 ± 2.2 vs 7.9 ± 0.8) originating from [14

C]glu-cose This suggests that Fru1,6P2 is strongly channeled

all the way to CO2, 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 CO2, although with varying degrees

of success Cells grown in 2% glycerol evolved 14CO2 from Fru1,6P2 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 CO2 from Fru6P (data not shown) However when 25 lm Fru6P was added to the growth medium, CO2 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 14CO2 evolution

of coincubating [14C]intermediates with [12C]glucose Whereas the [12C]glucose tended to decrease 14CO2 when [12C]glucose was coincubated with a [14 C]inter-mediate, the radioactivity in the CO2 evolved was significant compared to that in the absence of [12C]glucose; 42, 72, and 67% for [14C]Glu6P, [14C]Fru6P and [14C]Fru1,6P2, respectively This is not in conflict with the result that unlabeled Fru1,6P2 did not dilute the radioactivity in 14CO2 when it was coincubated with [14C]glucose (white bar vs black bar, Fig 2) Despite the fact that exogenous Fru1,6P2 entered the cell, it was unsuccessful in competing for the catalytic site of aldolase (EC 4.1.2.13) with the Fru1,6P2 just made in the pathway originating from [14C]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 [14C]Fru1,6P2 to 14CO2 in the presence of [12C]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 CO2via 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 CO2via 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

channeling were zero When the data from both figures

are combined, we are able to calculate the expectation

if the fraction channeled were zero (Fch¼ 0) When

Fch¼ 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

CO2, 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 [14C]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 [14C]glucose and unlabeled challenging intermediate (Glu6P, Fru6P, or Fru1,6P2) 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 with14C-labeled intermedi-ates (Glu6P, Fru6P or Fru1,6P2) 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).

[14C]Glucose is converted to [14C]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,

[14C]Fru6P will equilibrate with [12C]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 [12C]Fru6P had been

present To the degree that the 14 C and 12 C intermediates do not

equilibrate, the amount of 14 CO2evolved will be decreased to a

les-ser degree, unless downstream14C and 12C intermediates

equili-brate.

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 14CO2 from the earliest

time point (see Results) Also recall from the Results

that the CO2 evolved from [14C]glucose was strictly

proportional to the total uptake of glucose Thus, we

can use CO2 evolved as a reliable proxy for the total

uptake

To facilitate the following discussion, let A¼ the

14CO2evolved when cells were incubated with [14

C]glu-cose alone; B¼ the 14CO2 evolved when cells were

incubated with [14C]glucose plus [12C]exogenous

inter-mediate; C¼ the 14CO2 evolved when cells were

incu-bated with [14C]exogenous intermediate alone; D¼ the

14CO2 evolved when cells were incubated with

[14C]exogenous intermediate plus [12C]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 [14C]Fru6P converted to 14CO2

from [14C]glucose as the result of coincubation of

[14C]glucose and [12C]intermediate is proportional to

‘B’ as defined above Likewise the amount of

[14C]Fru6P converted to 14CO2 from exogenous

[14C]Fru6P when coincubated with [14C]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,

Fch¼ 0), the expected dilution of 14CO2 originating

from14C-labeled glucose when challenged by unlabeled

intermediate, is equal to B⁄ (B + D) That is, if

B⁄ A ¼ B ⁄ (B + D), then Fch¼ 0

When the exogenous intermediate is completely

unsuccessful in decreasing the radioactivity of the CO2

evolved from [14C]glucose, then

B=A¼ 1; and Fch¼ 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 Fch¼ 0

Likewise, when the intermediate made from glucose

does not dilute the radioactivity of CO2from a

coincu-bation of [12C]glucose and [14C]intermediate, then

D=C¼ 1; and Fch¼ 1

Values of Fch 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 14CO2 evolved from [14C]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 Fru1,6P2, there was a large, significant difference between the observed impact on 14CO2 evolution from [14C]glucose when coincubated with [12C]Fru1,6P2 (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 (Fch).

Fig 5 Expected fractional effect on 14 CO2evolved of coincubating

E coli cells with [14C]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.

[B⁄ (B + D)] (Fig 5) This result is consistent with the

Fru1,6P2 just made in the pathway being channeled

By contrast, for Glu6P 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 Fru1,6P2 to CO2 was channeled (Table 3;

Fch¼ 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 CO2 This is a surprising result

When the paradigm of Fig 4 is used to calculate Fch

for Glu6P or Fru6P just made in the pathway, the

results are Fch¼ 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 Fru1,6P2 to CO2 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 Fru1,6P2 to CO2 is consistent with the existence of a glycolytic complex that holds together long enough for the Fru1,6P2 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.

+25 l M Fru6P

22 m M glycerol

A (nmol 14 CO2evolved from incubation

with [ 14 C]glucose alone)

B (nmol 14 CO 2 evolved from incubation

with [14C]glucose plus unlabeled intermediate)

C (nmol 14 CO2evolved from incubation

with [ 14 C]intermediate alone)

D (nmol14CO 2 evolved from incubation

with [14 C]intermediate plus unlabeled glucose)

Evaluation of channeling of intermediate just made in the pathway

B ⁄ (B + D) (expected effect of unlabeled

challenger on 14 CO2evolution from

[ 14 C]glucose if no channelling

(P ¼ 0.005) Evaluation of channeling of exogenous intermediate

D ⁄ (B + D) (expected effect of unlabeled

challenger on 14 CO2evolution from

14 C-labeled intermediate if no channelling

the subsequent steps of glycolysis and on to CO2 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, Fru1,6P2 was

not strongly channeled in mouse L-929 cells

Our results show that, under certain conditions,

E coli cells channel an intermediate (Fru1,6P2) from

early in the glycolytic pathway all the way to CO2

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-14C]-, [1-14C]- and [6-14C]glucose, [U-14C]Glu6P, [U-14C]

Fru6P, and [U-14C]Fru1,6P2, 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 K2HPO4, 4.5 g KH2PO4, 1 g (NH4)2SO4, 0.5 g trisodium citrate, 10 mm MgSO4, 1 mm CaCl2, 5 mg 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 Mg2+

or Ca2+) such that the absorbance was
10 at 600 nm About 20 lL 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 with14C 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.

RK9117 D(argF-lac)U169 araD139 thi gyrA219 relA rpsL150 non polA1 D(ilvBN-uhpABCT ¢)2095 zig621::Tn10 uhpA + B + C 91::4 (Con)T +

Rk9118 D(argF-lac)U169 araD139 thi gyrA219 relA rpsL150 non polA1 D(ilvBN-uhpABCT ¢)2095 zig621::Tn10 uhpA +

B+C 91::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 lL H2O, 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

Mean CPM ± SE

Mean DPM ± SE

tions were terminated by adding 100 lL 70% (v⁄ v) HClO4.

The outgassed CO2was captured on the base impregnated

fil-ter paper Affil-ter incubation, the filfil-ter 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 CO2 production was calculated as

nmolsÆmin)1¼ nmol equivalents of14

C source· percentage conversion⁄ (100 · min)

The term ‘nmol equivalents of14C-source’ is included in

order to take into account the fact that six molecules of

CO2 were produced from one molecule of glucose, Glu6P,

Fru6P or Fru1,6P2 via glycolysis followed by mixed acid

fermentation By contrast, only one molecule of CO2 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 lL of

10% NaOH was present in samples used to measure CO2

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 CO2evolved

Because we were investigating channeling from early

glyco-lytic intermediates to pyruvate and on to CO2, any CO2

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 CO2 being evolved

from the C-1 position of glucose When glucose is

catabo-lized completely to CO2 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 CO2 Thus the

amount of CO2 produced in the OPPP is CO2 from

[1-14C]glucose minus CO2 from [6-14C]glucose The total

amount of CO2produced by the OPPP plus that produced

by the oxidation of pyruvate was calculated from

incuba-tions with [U-14C]glucose The ratio of the above two

values is the fraction of CO2evolved 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-14C]glucose (1 mm)

alone, (b) [U-14C]glucose (1 mm) plus 12C 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 Fru1,6P2) alone, and (d) [U-14 C]interme-diate (5 mm Glu6P or 5 mm Fru6P or 10 mm Fru1,6P2) plus [12C]glucose (1 mm)

Acknowledgements

This work was supported by an SGER NSF grant: NSD Grant # MCB-02004900

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