戊糖磷酸途径(PPP)的氧化阶段涉及葡萄糖-6-磷酸脱氢酶(G6PDH),6-磷酸葡萄糖酸内酯酶(6PGL),和6-磷酸葡萄糖酸脱氢酶(6PGDH),对细胞内NADPH的生成至关重要,这些酶催化葡萄糖-6-磷酸(G6P)转化为核酮糖-5-磷酸(Ribu5-P)。我们以前研究过氧自由基(ROO•)介导的大肠杆菌G6PDH的氧化失活,6PGL,6PGDH然而,这些数据是从每种酶独立暴露于ROO•的实验中获得的,一种不反映生物现实的状况。在这项工作中,我们研究了当这些酶共同暴露于ROO时,如何调节NADPH的产生。将酶混合物(1:1:1比例)暴露于由100mM2,2'-偶氮双(2-甲基丙脒)二盐酸盐(AAPH)热分解产生的ROO·。NADPH在340nm处定量,和蛋白质氧化通过具有质谱检测(LC-MS)的液相色谱进行分析。使用数学模型对获得的数据进行了合理化。非氧化的酶的混合物,G6P和NADP+产生约175μMNADPH。计算模拟显示与NADPH形成相关的G6P持续下降,与实验数据一致。当酶混合物暴露于AAPH(3小时,37ºC),检测到较低水平的NADPH(~100μM),这也符合计算模拟。LC-MS分析表明Tyr发生了变化,Trp,和Met残基,但浓度低于分离的酶检测到的浓度。NADPH生成的定量表明,在氧化的初始阶段,途径活性没有改变,与G6PDH对途径氧化阶段失活的缓冲作用一致。
The oxidative phase of the pentose phosphate pathway (PPP) involving the enzymes glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6PGL), and 6-phosphogluconate dehydrogenase (6PGDH), is critical to
NADPH generation within cells, with these enzymes catalyzing the conversion of glucose-6-phosphate (G6P) into ribulose-5-phosphate (Ribu5-P). We have previously studied peroxyl radical (ROO•) mediated oxidative inactivation of E. coli G6PDH, 6PGL, and 6PGDH. However, these data were obtained from experiments where each enzyme was independently exposed to ROO•, a condition not reflecting biological reality. In this work we investigated how
NADPH production is modulated when these enzymes are jointly exposed to ROO•. Enzyme mixtures (1:1:1 ratio) were exposed to ROO• produced from thermolysis of 100 mM 2,2\'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). NADPH was quantified at 340 nm, and protein oxidation analyzed by liquid chromatography with mass spectrometric detection (LC-MS). The data obtained were rationalized using a mathematical model. The mixture of non-oxidized enzymes, G6P and NADP+ generated ∼175 μM
NADPH. Computational simulations showed a constant decrease of G6P associated with
NADPH formation, consistent with experimental data. When the enzyme mixture was exposed to AAPH (3 h, 37 ºC), lower levels of
NADPH were detected (∼100 μM) which also fitted with computational simulations. LC-MS analyses indicated modifications at Tyr, Trp, and Met residues but at lower concentrations than detected for the isolated enzymes. Quantification of NADPH generation showed that the pathway activity was not altered during the initial stages of the oxidations, consistent with a buffering role of G6PDH towards inactivation of the oxidative phase of the pathway.