Brindle K M
Department of Biochemistry, University of Oxford, England.
Biochemistry. 1988 Aug 9;27(16):6187-96. doi: 10.1021/bi00416a054.
31P NMR magnetization-transfer measurements were used to measure flux between inorganic phosphate and ATP in the reactions catalyzed by phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase in anaerobic cells of the yeast Saccharomyces cerevisiae. Flux between ATP and Pi and glucose consumption and ethanol production were measured in cells expressing different levels of phosphoglycerate kinase activity. Overexpression of the enzyme was obtained by transforming the cells with a multicopy plasmid containing the phosphoglycerate kinase coding sequence and portions of the promoter element. Fluxes were also measured in cells in which the glyceraldehyde-3-phosphate dehydrogenase activity had been lowered by limited incubation with iodoacetate. These measurements showed that both enzymes have low flux control coefficients for glycolysis but that phosphoglycerate kinase has a relatively high flux control coefficient for the ATP----Pi exchange catalyzed by the two enzymes. The Pi----ATP exchange velocities observed in the cell were shown to be similar to those displayed by the isolated enzymes in vitro under conditions designed to mimic those in the cell with respect to the enzyme substrate concentrations.
利用³¹P核磁共振磁化转移测量技术，测定了酿酒酵母厌氧细胞中磷酸甘油酸激酶和甘油醛-3-磷酸脱氢酶催化反应中无机磷酸与ATP之间的通量。在表达不同水平磷酸甘油酸激酶活性的细胞中，测定了ATP与Pi之间的通量、葡萄糖消耗和乙醇生成。通过用含有磷酸甘油酸激酶编码序列和部分启动子元件的多拷贝质粒转化细胞，实现了该酶的过表达。还在与碘乙酸有限孵育而使甘油醛-3-磷酸脱氢酶活性降低的细胞中测量了通量。这些测量结果表明，两种酶对糖酵解的通量控制系数都较低，但磷酸甘油酸激酶对这两种酶催化的ATP----Pi交换具有相对较高的通量控制系数。结果表明，在细胞中观察到的Pi----ATP交换速度与在体外模拟细胞内酶底物浓度条件下分离的酶所显示的速度相似。
