IN VIVO MEASUREMENTS OF ENZYME CHANNELING

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。许多代谢途径中的中间体被从一种途径酶“引导”到另一种途径酶的假设被广泛接受，但不是普遍接受。它仍然存在争议的一个原因是，许多弱酶复合物在分离过程中被解离。通道可能适用的一个原因是，细胞中没有足够的水来支持所有途径中间体在KM的均匀浓度，这是传统上认为允许途径发挥作用所需的近似浓度。Srere假设存在“代谢物”，即途径酶的短暂关联，而不是稳定的复合物（例如，电子传递链的细胞色素复合物或色氨酸合成酶亚基的共价键）。据推测，序列酶的接近性会导致第一种酶的产物在竞争第二种酶的活性位点时，与散装培养基中相同物种的分子相比具有优势。也就是说，途径中间体与细胞内的相同分子不属于同一池。在该途径中产生的中间产物被“引导”到下一个酶。为了测试通道的前景，我们正在实施电喷雾质谱法结合离线离子交换色谱法来测量葡萄糖/半乳糖代谢过程中产物磷酸糖的相对丰度。使用同位素标记的起始材料，我们将能够看到含有同位素的产品是否被未标记的材料稀释（无通道）或保持未稀释（通道）。

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).