| Co-Investigator(Kenkyū-buntansha) |
SATO Hideaki Tohoku University, Institute for Chemical Reaction Science, Research Associate, 反応化学研究所, 助手 (60271996)
SAGAMI Ikuko Tohoku University, Institute for Chemical Reaction Science, Lecturer, 反応化学研究所, 講師 (10143033)
KONAMI Hideo Tohoku University, Institute for Chemical Reaction Science, Associate professor, 反応化学研究所, 助教授 (40186713)
ITO Osamu Tohoku University, Institute for Chemical Reaction Science, Professor, 反応化学研究所, 教授 (30006332)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1996: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1995: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
(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.
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