TAILIEUCHUNG - Báo cáo khoa học: Critical roles of Asp40 at the haem proximal side of haem-regulated phosphodiesterase from Escherichia coli in redox potential, auto-oxidation and catalytic control

In haem-regulated phosphodiesterase (PDE) fromEscheri-chia coli(EcDOS), haem is bound to the PAS domain, and the redox state of the haem iron regulates catalysis by the generatedmutants ofAsp40, which forms a hydrogen bondwithHis77 (a proximal haemaxial ligand) via twowater molecules, and a salt bridge withArg85 at the protein surface. The redox potential of haem was markedly increased from67 mVvs. the standardhydrogenelectrode in the wild-type enzyme to 95 mV and 114 mV in the Ala and Asn mutants, respectively. . | Eur. J. Biochem. 271 3937-3942 2004 FEBS 2004 doi Critical roles of Asp40 at the haem proximal side of haem-regulated phosphodiesterase from Escherichia coli in redox potential auto-oxidation and catalytic control Miki Watanabe Hirofumi Kurokawa Tokiko Yoshimura-Suzuki Ikuko Sagami and Toru Shimizu Institute of Multidisciplinary Research for Advanced Materials Tohoku University Sendai Japan 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 to and 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. Keywords auto-oxidation haem axial ligand haem sensor phosphodiesterase redox potential. Haem-regulated phosphodiesterase PDE from Escherichia coli Ec DOS is a haem sensor enzyme composed of two functional domains an N-terminal haem-bound sensor domain and a C-terminal PDE catalytic domain . Catalysis by this enzyme is regulated by the haem redox state in that PDE is functional in the Fe II haembound enzyme but not the Fe III haem-bound enzyme . The crystal structure of the haem-bound domain revealed a typical PAS .

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