Johnson E A, Levine R L, Lin E C
J Bacteriol. 1985 Oct;164(1):479-83. doi: 10.1128/jb.164.1.479-483.1985.
Anaerobically induced NAD-linked glycerol dehydrogenase of Klebsiella pneumoniae for fermentative glycerol utilization was reported previously to be inactivated in the cell during oxidative metabolism. In vitro inactivation was observed in this study by incubating the purified enzyme in the presence of O2, Fe2+, and ascorbate or dihydroxyfumarate. It appears that O2 and the reducing agent formed H2O2 and that H2O2 reacted with Fe2+ to generate an activated species of oxygen which attacked the enzyme. The in vitro-oxidized enzyme, like the in vivo-inactivated enzyme, showed an increased Km for NAD (but not glycerol) and could no longer be activated by Mn2+ which increased the Vmax of the native enzyme but decreased its apparent affinity for NAD. Ethanol dehydrogenase and 1,3-propanediol oxidoreductase, two enzymes with anaerobic function, also lost activity when the cells were incubated aerobically with glucose. However, glucose 6-phosphate dehydrogenase (NADP-linked), isocitrate dehydrogenase, and malate dehydrogenase, expected to function both aerobically and anaerobically, were not inactivated. Thus, oxidative modification of proteins in vivo might provide a mechanism for regulating the activities of some anaerobic enzymes.
先前报道，肺炎克雷伯菌厌氧诱导的与NAD相关的甘油脱氢酶用于发酵性甘油利用，在氧化代谢过程中会在细胞内失活。在本研究中，通过在O2、Fe2+以及抗坏血酸或二羟基富马酸存在的情况下孵育纯化的酶，观察到了体外失活现象。似乎O2和还原剂形成了H2O2，并且H2O2与Fe2+反应生成了一种活性氧物种，该物种攻击了酶。体外氧化的酶，与体内失活的酶一样，对NAD（而非甘油）的Km值增加，并且不再能被Mn2+激活，Mn2+可增加天然酶的Vmax，但会降低其对NAD的表观亲和力。乙醇脱氢酶和1,3 - 丙二醇氧化还原酶这两种具有厌氧功能的酶，当细胞与葡萄糖进行需氧孵育时也会失去活性。然而，预计在有氧和无氧条件下均起作用的6 - 磷酸葡萄糖脱氢酶（与NADP相关）、异柠檬酸脱氢酶和苹果酸脱氢酶并未失活。因此，体内蛋白质的氧化修饰可能为调节某些厌氧酶的活性提供一种机制。
