tailieunhanh - Báo cáo khoa học: Quantitative estimation of channeling from early glycolytic intermediates to CO2 in intact Escherichia coli

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. | iFEBS Journal 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 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@ Website http Received 30 December 2004 revised 31 March 2005 accepted 7 April 2005 doi 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 interaction 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 channeled 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 transformation to fructose-6-phosphate. The data also suggest that in addition to pathway enzymes being associated with one another some are

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