N ACETYLBENZIDINE PEROXYGENASE METABOLISM BY PROSTAGLANDIN H SYNTHASE

前列腺素H合成酶(PHS)催化合成前列腺素H2 导致该酶的双电子氧化。主动型减员 酶是通过减少被氧化的辅因子而再生的。 这份报告研究了公羊精液中的PHS的机制 囊泡微体，催化还原辅因子的氧化 N-乙酰基联苯胺(ABZ)。在将0.06 mm ABZ转换为ITS期间 最终产物4‘-硝基-4-乙酰氨基联苯，一种新的代谢物 当1 mM抗坏血酸与0.2 mM 花生四烯酸或0.5 mM H_2O_2为底物。这种代谢物 与合成的N‘-羟基-N-乙酰基联苯胺(N’Ha)共洗脱 N-羟基-N-乙酰基联苯胺。新的代谢物被鉴定为 ESI/MS/N‘Ha女士占总数的10% 经高效液相色谱检测放射性恢复正常。当N‘Ha取代ABZ时， PHS将N‘Ha代谢成4’-硝基-4-乙酰氨基联苯。抑制者 研究表明，新陈代谢 是由于PHS而不是细胞色素P-450所致。DMPO、甘露醇和超氧化物 歧化酶不会改变新陈代谢。摄氧量研究没有 演示对分子氧的需求。当[18O]双氧水 以~(18)O为底物，观察到了~(18)O的浓集 4‘-硝基-4-乙酰氨基-联苯，而不是N’Ha。97%的浓缩是 对一个18O原子观察到17+7%，对两个18O原子观察到17+7%。The Rapid 建议用18O-N‘Ha与水的交换来解释缺乏 N‘Ha的浓缩和两个~(18)O原子的低浓缩 4‘-硝基-4-乙酰氨基联苯。结果显示了一种过氧化物酶 PHS对ABZ和N‘Ha的氧化及对ABZ分步氧化的启示 N‘-羟基、4’-亚硝基和4‘-硝基产物。

Synthesis of prostaglandin H2 by prostaglandin H synthase (PHS) results in a two-electron oxidation of the enzyme. An active reduced enzyme is regenerated by reducing cofactors, which become oxidized. This report examines the mechanism by which PHS, from ram seminal vesicle microsomes, catalyzes the oxidation of the reducing cofactor N-acetylbenzidine (ABZ). During the conversion of 0.06 mM ABZ to its final end product 4'-nitro-4-acetylamino-biphenyl, a new metabolite was observed when 1 mM ascorbic acid was present with either 0.2 mM arachidonic acid or 0.5 mM H2O2 as substrate. This metabolite co-eluted with synthetic N'-hydroxy-N-acetylbenzidine (N'HA) and not N-hydroxy-N-acetylbenzidine. The new metabolite was identified as N'HA by ESI/MS/MS. N'HA represented as much as 10% of the total radioactivity recovered by HPLC. When N'HA was substituted for ABZ, PHS metabolized N'HA to 4'-nitro-4-acetylaminobiphenyl. Inhibitor studies demonstrated that metabolism was due t o PHS not cytochrome P-450. DMPO, mannitol, and superoxide dismutase did not alter metabolism. Oxygen uptake studies did not demonstrate a requirement for molecular oxygen. When [18O]H2O2 was used as substrate, 18O enrichment was observed for 4'-nitro-4-acetylamino-biphenyl, but not N'HA. A 97% enrichment was observed for one atom of 18O and 17 + 7% for two 18O atoms. The rapid exchange of 18O-N'HA with water was suggested to explain the lack of enrichment of N'HA and the low enrichment of two 18O atoms into 4'-nitro-4-acetylaminobiphenyl. Results demonstrate a peroxy-genase oxidation of ABZ and N'HA by PHS and suggest stepwise oxidation of ABZ to N'-hydroxy, 4'-nitroso, and 4'-nitro products.