Critical roles of Asp40 at the haem proximal side of haem-regulated phosphodiesterase from Escherichia coli in redox potential, auto-oxidation and catalytic control.
Critical roles of Asp40 at the haem proximal side of haem-regulated phosphodiesterase from Escherichia coli in redox potential, auto-oxidation and catalytic control.
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DOI：
10.1111/j.1432-1033.2004.04331.x
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发表时间：
2004-10
期刊：
影响因子：
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通讯作者：
Miki Watanabe;H. Kurokawa;Tokiko Yoshimura-Suzuki;I. Sagami;Toru Shimizu
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文献类型：
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作者：
Miki Watanabe;H. Kurokawa;Tokiko Yoshimura-Suzuki;I. Sagami;Toru Shimizu
在大肠杆菌（Ec DOS）的血液调节磷酸二酯酶（PDE）中，血红素与PAS结构域结合，血红素铁的氧化还原状态调节PDE结构域的催化作用。我们产生了Asp40突变体，它通过两个水分子与His77（近血红素轴向配体）形成氢键，并在蛋白质表面与Arg85形成盐桥。与标准氢电极相比，野生型酶的血红素氧化还原电位从67 mV显著增加到Ala和Asn突变体的95 mV和114 mV。此外，Ec DOS PAS的自氧化速率分别从0.0053 min和0.051 min显著提高到0.033 min（-1）。有趣的是，Asp40突变体的催化活性被完全消除。因此，Asp40似乎在血红素铁的电子结构和PDE结构域的氧化还原依赖的催化控制中发挥关键作用。本文从Asp40突变体的理化特性出发，探讨了催化控制Ec DOS的机理。
In haem-regulated phosphodiesterase (PDE) from Escherichia coli (Ec DOS), haem is bound to the PAS domain, and the redox state of the haem iron regulates catalysis by the PDE domain. We generated mutants of Asp40, which forms a hydrogen bond with His77 (a proximal haem axial ligand) via two water molecules, and a salt bridge with Arg85 at the protein surface. The redox potential of haem was markedly increased from 67 mV vs. the standard hydrogen electrode in the wild-type enzyme to 95 mV and 114 mV in the Ala and Asn mutants, respectively. Additionally, the auto-oxidation rate of Ec DOS PAS was significantly increased from 0.0053 to 0.051 and 0.033 min(-1), respectively. Interestingly, the catalytic activities of the Asp40 mutants were abolished completely. Thus, Asp40 appears to play a critical role in the electronic structure of the haem iron and redox-dependent catalytic control of the PDE domain. In this report, we discuss the mechanism of catalytic control of Ec DOS, based on the physico-chemical characteristics of the Asp40 mutants.