IN VIVO MEASUREMENTS OF ENZYME CHANNELING

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The hypothesis that intermediates in many metabolic pathways are "channeled" from one pathway enzyme to the next is widely, but not universally, accepted. One reason it remains controversial is that many of the weak enzyme complexes are dissociated during isolation. One reason that channeling may apply is that there is not enough water in the cell to support uniform concentrations of all pathway intermediates at KM, the approximate concentration traditionally assumed required to permit pathways to function. Srere postulated the existence of "metabolons," transient associations of pathway enzymes as opposed to stable complexes (e.g. cytochrome complexes of the electron transport chain or the covalent linkage of tryptophan synthase subunits). It is postulated that the proximity of sequential enzymes would cause the product of the first enzyme to have an advantage in competition for the active site of the second enzyme when compared to molecules of the same species within the bulk medium. That is, pathway intermediates 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. To test the prospect of channeling, we are implementing an electrospray mass spectrometry method coupled with off-line ion exchange chromatography to measure the relative abundances of sugar phosphates that are products in the glucose/galactose metabolism process. Using isotope labeled starting materials, we will be able to see whether the products containing the isotopes are diluted with unlabeled materials (no channeling) or remain undiluted (channeling).