Tài liệu Báo cáo khoa học: Quantitative estimation of channeling from early glycolytic intermediates

Quantitative estimation of channeling from early glycolytic

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: [email protected]

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 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

Fru1,6P2, fructose-1,6-bis phosphate; Fru6P, fructose-6-phosphate; Fch, 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