Inactivation of pyruvate formate-lyase by dioxygen: defining the mechanistic interplay of glycine 734 and cysteine 419 by rapid freeze-quench EPR.
Inactivation of pyruvate formate-lyase by dioxygen: defining the mechanistic interplay of glycine 734 and cysteine 419 by rapid freeze-quench EPR.
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双氧灭活丙酮酸甲酸裂解酶：通过快速冷冻淬灭 EPR 定义甘氨酸 734 和半胱氨酸 419 的机械相互作用。
DOI：
10.1021/bi002589k
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Kozarich,JW
中科院分区：
文献类型：
--
作者：
Zhang,W;Wong,KK;Magliozzo,RS;Kozarich,JW
来自大肠杆菌的丙酮酸甲酸裂解酶（PFL）催化丙酮酸和CoA可逆厌氧转化为乙酰CoA和甲酸。2.3.1.54活性PFL在G734处含有一个新的α-碳中心甘氨酰自由基，这是其催化活性所必需的。两个相邻的半胱氨酸残基，C418和C419，是必不可少的PFL活性，根据定点诱变研究。在暴露于空气时，活性PFL失去其活性，伴随着甘氨酰自由基的损失。先前对PFL的分子氧失活的EPR研究揭示了在手动混合和淬灭过程中基于蛋白质的过氧基和亚磺酰基自由基[Reddy et al.（1998）Biochemistry 37，558−563]。为了探讨这一过程的机制，我们进行了实验，使用快速冷冻淬火EPR光谱。在活性野生型或C418 A PFL与含氧溶液混合后，在最早的时间点（10 ms）出现短寿命的自由基中间体，随后出现长寿命的亚磺酰基自由基。这个短寿命自由基（g= 2.034，2.007）的轴向EPR谱是过氧自由基的特征。当C419 A PFL或双突变体[C418 A/C419 A] PFL与含氧溶液混合时，在10 ms时也观察到过氧化氢自由基，但在这种情况下持续超过12 s。这些观察结果提供了令人信服的证据，以支持提出的机制，其中分子氧淬灭活性酶中的甘氨酰自由基，所得的过氧自由基可能进一步与C419残基的巯基反应，形成亚磺酰基自由基。
Pyruvate formate-lyase fromEscherichia coli(EC 2.3.1.54; PFL) catalyzes the reversible anaerobic conversion of pyruvate and CoA into acetyl-CoA and formate. Active PFL contains a novel α-carbon centered glycyl radical at G734 that is required for its catalytic activity. Two adjacent cysteine residues, C418 and C419, are essential for PFL activity according to site-directed mutagenesis studies. Upon exposure to air, active PFL loses its activity with the concomitant loss of the glycyl radical. Previous EPR studies of dioxygen inactivation of PFL revealed protein-based peroxyl and sulfinyl radicals during the manual mixing and quenching process [Reddy et al. (1998)Biochemistry 37, 558−563]. To probe the mechanism of this process, we carried out experiments using rapid freeze-quench EPR spectroscopy. Upon mixing of active wild type or C418A PFL with oxygenated solution, a short-lived radical intermediate appears at the earliest time point (10 ms), followed by the appearance of a long-lived sulfinyl radical. The axial EPR spectrum of this short-lived radical (g= 2.034, 2.007) is characteristic of a peroxyl radical. When C419A PFL or the double mutant [C418A/C419A] PFL was mixed with oxygenated solution, the peroxyl radical was also observed at 10 ms but in this case persisted over 12 s. These observations provide compelling evidence to support a proposed mechanism in which dioxygen quenches the glycyl radical in the active enzyme and the resulting peroxyl radical may react further with the sulfhydryl group of the C419 residue to form the sulfinyl radical.