Benevolensky S V, Clifton D, Fraenkel D G
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 1994 Feb 18;269(7):4878-82.
Comparison of microbial strains with normal and high content of single enzymes is coming into use for metabolic analysis and in vivo assessment of enzyme function. We present an example for phosphoglucose isomerase and glucose metabolism in the yeast Saccharomyces cerevisiae. We use cell suspensions in conditions of inhibited protein synthesis and respiration, with low assimilation, rapid and linear glucose utilization, fermentation almost quantitative, and high enough cell density for direct preparation of extracts for metabolite analysis. The mass action ratio and fitting of fructose-6-P and glucose-6-P concentrations and kinetic parameters of the enzyme are not inconsistent with near equilibrium of the reaction in the wild-type strain and small if any change in the high level strain. However, this conclusion would require that the Vmax values underestimate the activity in the cell. On the other hand, the specific activities of glucose-6-P and fructose-1,6-P2 during metabolism of [2-3H]glucose are quite high which, together with knowledge of tritium exchange and isotope effects for the reaction in vitro, would point to the reaction in the wild-type strain being far from equilibrium; the specific activities are lower in the high level strain, indicating that extra enzyme is functional. One way to reconcile the latter results would be for tritium exchange to be considerably lower in vivo than known in vitro.
将单酶含量正常和高的微生物菌株进行比较，正被用于代谢分析和酶功能的体内评估。我们给出了酿酒酵母中磷酸葡萄糖异构酶和葡萄糖代谢的一个例子。我们使用处于蛋白质合成和呼吸受抑制、同化作用低、葡萄糖利用快速且呈线性、发酵几乎完全定量以及细胞密度足够高以便直接制备用于代谢物分析的提取物的条件下的细胞悬浮液。果糖 -6-磷酸和葡萄糖 -6-磷酸浓度的质量作用比及拟合情况以及该酶的动力学参数，与野生型菌株中反应接近平衡且高表达菌株中变化很小（如果有的话）是相符的。然而，这个结论需要Vmax值低估细胞中的活性。另一方面，在[2-³H]葡萄糖代谢过程中葡萄糖 -6-磷酸和果糖 -1,6-二磷酸的比活性相当高，结合体外反应的氚交换和同位素效应的知识，这表明野生型菌株中的反应远非平衡状态；高表达菌株中的比活性较低，表明额外的酶具有功能。协调后一种结果的一种方法可能是体内的氚交换比体外已知的要低得多。
