Roles for the two cysteine residues of AhpC in catalysis of peroxide reduction by alkyl hydroperoxide reductase from Salmonella typhimurium
Roles for the two cysteine residues of AhpC in catalysis of peroxide reduction by alkyl hydroperoxide reductase from Salmonella typhimurium
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DOI：
10.1021/bi9713658
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发表时间：
1997-10-28
期刊：
影响因子：
2.9
通讯作者：
Poole, LB
中科院分区：
文献类型：
--
作者：
Ellis, HR;Poole, LB
半胱氨酸残基Cys 46和Cys 165，形成亚基间二硫键的烷基过氧化氢还原酶（AhpR）系统从鼠伤寒沙门氏菌的过氧化物AhpC蛋白的催化性能进行了研究。AhpR系统由AhpC和黄素蛋白还原酶AhpF组成，催化有机氢过氧化物和过氧化氢的吡啶核苷酸依赖性还原。含二硫键的胰蛋白酶肽的氨基酸序列分析表明，存在两个相同的二硫键，每个二聚体的氧化AhpC位于Cys 46上的一个亚基和Cys 165上的其他之间。突变AhpC蛋白只含有一个（C46和C165 S）或没有（C46，165 S）半胱氨酸残基进行纯化，并显示圆二色性研究，表现出没有重大的破坏二级结构。在NADH依赖的过氧化物酶测定中，在AhpF的存在下，C165 S突变体是完全活跃的野生型AhpC相比，而C46 S和C46，165 S显示没有过氧化物酶活性。此外，只有C165 S被1当量的过氧化氢氧化，得到在催化量的AhpF存在下可被NADH化学计量还原的物质。氧化的C165 S还与化学计量的含硫醇试剂2-硝基-5-硫代苯甲酸快速反应，生成混合二硫化物，并且易于被过氧化氢灭活，强烈支持其鉴定为半胱氨酸次磺酸（Cys 46-SOH）。C46 S突变体对过氧化物的反应性的缺乏不是剩余硫醇不可及的结果，如通过其用5，5 '-二硫代双（2-硝基苯甲酸）的修饰所证明的，而是可能是由于缺乏近端活性位点碱，该近端活性位点碱将通过质子供给到差的RO-离去基团来支持催化。我们的研究结果清楚地表明，Cys 46作为AhpC的过氧化物中心，Cys 165作为一个重要的残基，通过与氧化蛋白（Cys 46-SOH）的新生次磺酸反应，产生一个稳定的二硫键，从而防止Cys 46-SOH被底物进一步氧化，以保持野生型AhpC的活性。
The catalytic properties of cysteine residues Cys46 and Cys165, which form intersubunit disulfide bonds in the peroxidatic AhpC protein of the alkyl hydroperoxide reductase (AhpR) system from Salmonella typhimurium, have been investigated. The AhpR system, composed of AhpC and a flavoprotein reductase, AhpF, catalyzes the pyridine nucleotide-dependent reduction of organic hydroperoxides and hydrogen peroxide. Amino acid sequence analysis of the disulfide-containing tryptic peptide demonstrated the presence of two identical disulfide bonds per dimer of oxidized AhpC located between Cys46 on one subunit and Cys165 on the other. Mutant AhpC proteins containing only one (C46S and C165S) or no (C46,165S) cysteine residues were purified and shown by circular dichroism studies to exhibit no major disruptions in secondary structure. In NADH-dependent peroxidase assays in the presence of AhpF, the C165S mutant was fully active in comparison with wild-type AhpC, while C46S and C46,165S displayed no peroxidatic activity. In addition, only C165S was oxidized by 1 equiv of hydrogen peroxide, giving a species that was stoichiometrically reducible by NADH in the presence of a catalytic amount of AhpF. Oxidized C165S also reacted rapidly with a stoichiometric amount of the thiol-containing reagent 2-nitro-5-thiobenzoic acid to generate a mixed disulfide, and was susceptible to inactivation by hydrogen peroxide, strongly supporting its identification as a cysteine sulfenic acid (Cys46-SOH). The lack of reactivity of the C46S mutant toward peroxides was not a result of inaccessibility of the remaining thiol as demonstrated by its modification with 5,5'-dithiobis(2-nitrobenzoic acid), but could be due to the lack of a proximal active-site base which would support catalysis through proton donation to the poor RO- leaving group. Our results clearly identify Cys46 as the peroxidatic center of AhpC and Cys165 as an important residue for preserving the activity of wild-type AhpC by reacting with the nascent sulfenic acid of the oxidized protein (Cys46-SOH) to generate a stable disulfide bond, thus preventing further oxidation of Cys46-SOH by substrate.