Kinetic and mutational studies of three NifS homologs from Escherichia coli:: Mechanistic difference between L-cysteine desulfurase and L-selenocysteine lyase reaction
Kinetic and mutational studies of three NifS homologs from Escherichia coli:: Mechanistic difference between L-cysteine desulfurase and L-selenocysteine lyase reaction
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DOI：
10.1093/oxfordjournals.jbchem.a022641
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发表时间：
2000-04-01
影响因子：
2.7
通讯作者：
Esaki, N
中科院分区：
文献类型：
--
作者：
Mihara, H;Kurihara, T;Esaki, N
我们已经从大肠杆菌，CSD，CsdB，和IscS，这似乎是参与铁硫簇的形成和/或硒磷酸盐的生物合成纯化三个NifS同系物。所有三种同系物分别催化Se和S从L-硒代半胱氨酸和L-半胱氨酸中消除，形成L-丙氨酸。这些吡哆醛5 '-磷酸酶通过失败的转氨作用失活，产生丙酮酸和吡哆胺5'-磷酸形式的酶。当加入丙酮酸时，它们对L-硒代半胱氨酸而不是L-半胱氨酸表现出Michaelis-Menten行为，丙酮酸显著增强CSD对L-硒代半胱氨酸的活性，令人惊讶的是，丙酮酸对L-半胱氨酸的酶活性没有增加那么多，提示L-半胱氨酸脱硫和L-硒代半胱氨酸降解存在不同的限速步骤或反应机理。我们用Ala取代CSD中的Cys 358、CsdB中的Cys 364和IscS中的Cys 328中的每一个，这些残基对应于棕色固氮菌NifS的催化必需的Cys 325。对L-半胱氨酸的酶活性几乎完全消除了突变，而对L-硒代半胱氨酸的活性影响小得多。这表明L-半胱氨酸脱硫的反应机理与L-硒代半胱氨酸分解的反应机理不同，保守的半胱氨酸残基仅在L-半胱氨酸脱硫中起关键作用。
We have purified three NifS homologs from Escherichia coli, CSD, CsdB, and IscS, that appear to be involved in iron-sulfur cluster formation and/or the biosynthesis of selenophosphate. All three homologs catalyze the elimination of Se and S from L-selenocysteine and L-cysteine, respectively, to form L-alanine, These pyridoxal 5'-phosphate enzymes were inactivated by abortive transamination, yielding pyruvate and a pyridoxamine 5'-phosphate form of the enzyme. The enzymes showed non-Michaelis-Menten behavior for L-selenocysteine and L-cysteine, When pyruvate was added, they showed Michaelis-Menten behavior for L-selenocysteine but not for L-cysteine, Pyruvate significantly enhanced the activity of CSD toward L-selenocysteine, Surprisingly, the enzyme activity toward L-cysteine was not increased as much by pyruvate, suggesting the presence of different rate-limiting steps or reaction mechanisms for L-cysteine desulfurization and the degradation of L-selenocysteine. We substituted Ala for each of Cys358 in CSD, Cys364 in CsdB, and Cys328 in IscS, residues that correspond to the catalytically essential Cys325 of Azotobacter vinelandii NifS. The enzyme activity toward L-cysteine was almost completely abolished by the mutations, whereas the activity toward L-selenocysteine was much less affected. This indicates that the reaction mechanism of L-cysteine desulfurization is different from that of L-selenocysteine decomposition, and that the conserved cysteine residues play a critical role only in L-cysteine desulfurization.