SGER: In Vivo Measurements of Channeling

许多代谢途径中的中间产物被从一种途径的酶“引导”到下一种途径的假说被广泛接受，但并非普遍接受。通道的机制被推定为途径酶的瞬时联系。据推测，顺序酶的接近会使第一种酶的产物在竞争第二种酶的活性部位时比在散装介质中的同类分子具有优势。另一种方法是，途径中间产物不是细胞内相同分子的同一池的一部分，途径中产生的中间产物被“引导”到下一种酶。对这一假说的研究通常集中在假定的机制上；即，顺序途径酶的关联。采用的方法包括几种层析技术、共沉淀和共纯化。虽然阳性结果是有提示意义的，但它们并不是决定性的，因为即使这些酶是相关的，通道也不需要发生。其他方法，包括同位素稀释实验，经常支持通道的存在，但在体外系统中，不能很好地代表拥挤的、有结构的活细胞环境。体内实验中采用的方法包括遗传学方法、核磁共振和同位素稀释。虽然这些实验的一些报告强烈支持通灵的存在，但这些技术的使用只允许对通灵进行定性评估。这里提出的工作应该导致对体内功能系统中的通道进行定量评估。为了简单起见，考虑一个两种酶的途径。底物A与酶1结合形成中间体B。B是酶2的底物。产物是C。如果可以诱导细胞摄取B*(星号表示B具有不同的稳定同位素，如13C，与B不同)，而B*作为酶2的底物，则产物为C*。如果没有信道化，则B*/B=C*/C。如果存在100%信道化，则C*=0。在已知比率B*/B和C*/C的情况下，可以计算出导流的量化值。目前使用的其他方法都不能提供这一信息。适当的比例将使用与适当柱接口的质谱仪(MS)来测量。葡萄糖-6-磷酸(G6P)的代谢是这一提议的焦点之一。己糖激酶产生的G6P是否被引导到磷酸戊糖途径氧化支链的下一个酶，G6P脱氢酶将被研究。适当的酵母突变株在葡萄糖(GLC)存在的情况下会吸收半乳糖(GAL)。经过四个步骤，GAL转化为G6P，G6P被氧化成6-磷酸葡萄糖(6PG)，酵母将在13C-GLC和12C-GAL中培养。将测量13C-G6P/12C-G6P和13C-GAL/12C-GAL的比率，并对结果进行如上所述的解释。G6P和6PG有异构体，如果与感兴趣的化合物同时进入MS，就会得到人工合成的结果。为了避免这种情况，异构体将在Dionex柱上离线分离。如有必要，将利用MS/MS技术对任何异构体杂质进行校正。将用原核生物E.Coli进行类似的实验，以研究醛缩酶和3-磷酸甘油醛脱氢酶之间的通道。有了这种方法，不仅可以量化导流的程度，而且还可以评估导流随着环境条件或营养状态的变化而发生的变化，以确定其对指导代谢物流动的可能贡献。

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.