SGER: In Vivo Measurements of Channeling

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The hypothesis that intermediates in many metabolic pathways are "channeled" from onepathway enzyme to the next is widely, but not universally, accepted. The mechanism ofchanneling is presumed to be the transient associations of pathway enzymes. It is postulated thatthe proximity of sequential enzymes would cause the product of the first enzyme to have anadvantage in competition for the active site of the second enzyme when compared to moleculesof the same species within the bulk medium. An alternative formulation is that pathwayintermediates 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. Investigations ofthis hypothesis often center on the presumed mechanism; i.e., the association of sequentialpathway enzymes. Methods employed include several chromatographic techniques, co-precipitation and co-purification. While positive results are suggestive, they are not conclusivesince channeling need not occur even if the enzymes are associated. Other approaches, includingisotope dilution experiments, have often supported the existence of channeling, but in vitrosystems are a poor proxy for the crowded, structured environment of the living cell. Approachesemployed in in vivo experiments include genetic approaches, NMR and isotope dilution. Whilesome of the reports from these experiments strongly support the existence of channeling, use ofthese techniques allow only a qualitative assessment of channeling. The work proposed hereshould lead to a quantitative evaluation of channeling in an in vivo, functioning system.For simplicity, consider a 2-enzyme pathway. Substrate A binds to Enzyme 1 to makeintermediate B. B is the substrate for Enzyme 2. The product is C. If the cells can be induced totake up B* (the asterisk indicating B has a different stable isotope, say 13 C, than does B), and B*serves as the substrate for Enzyme 2, then the product will be C*. If there is no channeling, thenB*/B = C*/C. If there is 100% channeling, then C* = 0. Given knowledge of the ratios B*/B &C*/C, quantitative values for channeling can be calculated. No other method presently in use canprovide this information. The appropriate ratios will be measured using a mass spectrometer (MS) interfaced with an appropriate column. The metabolism of glucose-6-phosphate (G6P) is one of the foci of this proposal. Whether G6P produced by hexokinase is channeled to the next enzyme in the oxidative limb of the pentose phosphate pathway, G6P dehydrogenase will be investigated. The appropriate mutant of yeast will take up galactose (gal) in the presence of glucose (glc). In four steps, gal is converted to G6P, which is oxidized to 6-phosphoguconate (6PG) Yeast will be incubated in 13 C-glc and 12 C-gal. The ratios 13 C-G6P / 12 C-G6P and 13 C-gal / 12 C-gal will be measured and the results interpreted as described above. G6P and 6PG have isomers which, if entered into the MS at the same time as the compounds of interest, would give artifactual results. To avoid this, the isomers will be separated on a Dionex column off-line. If necessary corrections for any isomeric impurity using MS/MS techniques will be made.An analogous experiment will be done with a prokaryote, E. coli, to investigate channelingbetween aldolase and glyceraldehyde-3-phosphate dehydrogenase. With this method in place not only can the degree of channeling be quantified, but also changes in channeling in response to changing environmental conditions or nutritional states may be evaluated for its possible contribution to directing the flow of metabolites.