权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Structure-Function Relationship of Biodeseigned NO Synthase and Dynamics of NO

生物设计的 NO 合成酶的结构-功能关系和 NO 动力学

基本信息

批准号：
07680670
负责人：
SHIMIZU Toru
金额：
$ 1.41万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

(1)Nitric oxide synthase (NOS) has a thiolate-coordinated heme active site similar to that of cytochrome P450 (P450). In the present study, NO bindings to cytochrome P450 1A2 (P450 1A2) distal mutants were studied in the presence of various substrates. We found that a mutation at Glu318 to Ala in the putative distal site of P450 1A2, suggested to be important in the O_2 activation of P450 reactions, markedly facilitates the reduction of the NO-ferric complex. Addition of 1,2 : 3,4-dibenzanthracene or phenanthrene almost abolished the mutation effect on the NO complex. Based on these results, together with other spectral and kinetics data, it is suggested that the NO-ferric complex stability of P450, and perhaps of NOS,is largely ascribed to an ionic bridge between NO and the distal carboxyl group.(2)We examined NO synthesis capability of rat liver cytochrome P450 1A2 (P450 1A2) from N^G-hydroxy-L-Arg (NHA) with both the peroxide-supported shunt system and the reconstituted system composed of P450 1A2 and the reductase. Roles of distal amino acids of P450 1A2 in the catalytic functions were also studied. No was synthesized effectively with the shunt reactions with k_<cat>=0.6-1.2nmol/nmolP450/min. NO was formed from NHA with the reductase alone, as well as, with the reconstituted system with turnover numbers of 26 and 62 pmol/nmolP450/min, respectively. A Glu318Ala mutation of P450 1A2 enhanced the shuntreaction activity up to 7.3-fold, whereas the mutation abolished the activity with the reconstituted system. Catalase markedly inhibited the activity in the reconstituted system, whereas it enhanced the shunt activity up to 2.2-fold. Superoxide dismutase and (6R)-5,6,7,8-tetrahydro-L-biopterin, which markedly enhance NO synthesis with NOS,strongly inhibited the NO synthesis in both the reconstituted and shunt systems.

(1)一氧化氮合酶(NOS)具有与细胞色素P450(P450)相似的硫醇配位的血红素活性位点。在本研究中，在各种底物存在的情况下研究了 NO 与细胞色素 P450 1A2 (P450 1A2) 远端突变体的结合。 We found that a mutation at Glu318 to Ala in the putative distal site of P450 1A2, suggested to be important in the O_2 activation of P450 reactions, markedly facilitates the reduction of the NO-ferric complex.添加1,2:3,4-二苯并蒽或菲几乎消除了对NO复合物的突变作用。 Based on these results, together with other spectral and kinetics data, it is suggested that the NO-ferric complex stability of P450, and perhaps of NOS,is largely ascribed to an ionic bridge between NO and the distal carboxyl group.(2)We examined NO synthesis capability of rat liver cytochrome P450 1A2 (P450 1A2) from N^G-hydroxy-L-Arg (NHA) 具有过氧化物支持的分流系统和由 P450 1A2 和还原酶组成的重构系统。还研究了 P450 1A2 远端氨基酸在催化功能中的作用。通过k_<cat>=0.6-1.2nmol/nmolP450/min的分流反应有效地合成了No。 NO was formed from NHA with the reductase alone, as well as, with the reconstituted system with turnover numbers of 26 and 62 pmol/nmolP450/min, respectively. A Glu318Ala mutation of P450 1A2 enhanced the shuntreaction activity up to 7.3-fold, whereas the mutation abolished the activity with the reconstituted system.过氧化氢酶显着抑制重建系统中的活性，而将分流活性增强至 2.2 倍。 Superoxide dismutase and (6R)-5,6,7,8-tetrahydro-L-biopterin, which markedly enhance NO synthesis with NOS,strongly inhibited the NO synthesis in both the reconstituted and shunt systems.

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

中野亮介: "Tris (2, 2´-bipyridyl) ruthenium (II) -Mediated Photoinduced Electron Transfer of Engineered Cytochrome P450 1A2" Journal of Photobiochemistry and Photobiology. 100(発売予定). (1996)

Ryosuke Nakano：“Tris (2, 2´-bipyridyl) ruthenium (II) -Mediated Photoinduced Electron Transfer of Engineered Cytochrome P450 1A2”《光生物化学和光生物学杂志》100（待发布）。