Analysis of the kinetic and redox properties of the NADH peroxidase R303M mutant: correlation with the crystal structure.
Analysis of the kinetic and redox properties of the NADH peroxidase R303M mutant: correlation with the crystal structure.
复制标题
NADH 过氧化物酶 R303M 突变体的动力学和氧化还原特性分析：与晶体结构的相关性。
DOI：
10.1021/bi000553m
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发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
Claiborne,A
中科院分区：
文献类型：
--
作者：
Crane3rd,EJ;Yeh,JI;Luba,J;Claiborne,A
黄素蛋白NADH过氧化物酶的晶体结构表明，Arg 303侧链与活性位点His 10咪唑形成氢键，因此可能影响催化机制。R303 M突变体[E（FAD，Cys 42-次磺酸）]的连二亚硫酸盐滴定产生了具有增强的黄素荧光的双电子还原中间体（EH 2），并且在pH 7.0下几乎没有电荷转移吸光度;与野生型EH 2 pKaof ≤4.5相比，新生Cys 42-SH的pKa增加了3.5个单位以上。突变型过氧化物酶的NADH滴定产生相同的EH 2中间体，但与野生型酶的行为相反，该物种可直接还原为EH 4·NAD+复合物。动力学分析表明，R303 M突变体是严重受损，虽然活性，与kcat = 3 s-1在pH 7.0，5 °C;酶监测的周转结果表明，稳态主要由E-FADH 2·NAD+物种。当氧化的突变体与0.9当量的NADH/FAD厌氧反应时，在450 nm处观察到明显的两相模式;相对快速的黄素还原之后是2.6−2.7 s-1的再氧化（Rekkcat）。因此，用Met替换Arg 303导致改变的过氧化物酶形式，其中周转中的限速步骤是电子从FADH 2 → Cys 42-SOH的分子内转移。R303 M过氧化物酶的晶体结构已在2.45 μ m分辨率下进行了细化。除了消除Arg 303与His 10和Glu 14的相互作用外，突变体还表现出Cys 42-SOH侧链构象的显著变化，特别是相对于FAD和His 10。这些和其他结果提供了一个详细的了解Arg 303和它的作用，这种独特的黄素蛋白过氧化物酶的结构和机制。
The crystal structure of the flavoprotein NADH peroxidase shows that the Arg303 side chain forms a hydrogen bond with the active-site His10 imidazole and is therefore likely to influence the catalytic mechanism. Dithionite titration of an R303M mutant [E(FAD, Cys42-sulfenic acid)] yields a two-electron reduced intermediate (EH2) with enhanced flavin fluorescence and almost no charge-transfer absorbance at pH 7.0; the pKafor the nascent Cys42-SH is increased by over 3.5 units in comparison with the wild-type EH2pKaof ≤4.5. NADH titration of the mutant peroxidase yields the same EH2intermediate, but in contrast to the behavior of wild-type enzyme, this species can be reduced directly to an EH4·NAD+complex. Kinetic analyses demonstrate that the R303M mutant is severely compromised, although active, withkcat= 3 s-1at pH 7.0, 5 °C; enzyme-monitored turnover results indicate that the steady-state consists predominantly of an E-FADH2·NAD+species. When the oxidized mutant is reacted anaerobically with 0.9 equiv of NADH/FAD, a clearly biphasic pattern is observed at 450 nm; relatively rapid flavin reduction is followed by reoxidation at 2.6−2.7 s-1(∼kcat). Thus replacement of Arg303 with Met leads to an altered peroxidase form in which the rate-limiting step in turnover is the intramolecular transfer of electrons from FADH2→ Cys42-SOH. The crystal structure of the R303M peroxidase has been refined at 2.45 Å resolution. In addition to eliminating the Arg303 interactions with His10 and Glu14, the mutant exhibits a significant change in the conformation of the Cys42-SOH side chain relative to FAD and His10 in particular. These and other results provide a detailed understanding of Arg303 and its role in the structure and mechanism of this unique flavoprotein peroxidase.