N ACETYLBENZIDINE PEROXYGENASE METABOLISM BY PROSTAGLANDIN H SYNTHASE

通过前列腺素H合酶（PHS）合成前列腺素H2 导致酶的双电子氧化。 一种活性还原剂 酶通过还原被氧化的辅因子而再生。 本报告探讨了机制，其中小灵通，从公羊精液 囊泡微粒体，催化还原辅因子的氧化 N-乙酰联苯胺（ABZ）。 在将0.06 mM ABZ转化为其 最终产物4 '-硝基-4-乙酰氨基-联苯，一种新的代谢产物 当1 mM抗坏血酸与0.2 mM 花生四烯酸或0.5mM H2 O2作为底物。 该代谢物 与合成的N '-羟基-N-乙酰基联苯胺（N' HA）共洗脱，而不 N-羟基-N-乙酰联苯胺。 新代谢产物被鉴定为 通过ESI/MS/MS测定N 'HA，N' HA占总量的10 通过HPLC回收的放射性。 当N 'HA取代ABZ时， PHS将N 'HA代谢为4'-硝基-4-乙酰氨基联苯。 抑制剂 研究表明新陈代谢 不是细胞色素P-450。 DMPO、甘露醇和超氧化物 歧化酶不改变代谢。 氧摄取研究没有 需要分子氧 当[18 O] H2 O2 作为底物， 4 '-硝基-4-乙酰氨基-联苯，但不是N' HA。 97%的浓缩是 观察到一个18 O原子和17 ± 7%的两个18 O原子。 快速 18 O-N 'HA与水的交换被认为可以解释 N 'HA的富集和两个18 O原子的低富集成 4 '-硝基-4-乙酰氨基联苯。 结果表明， PHS对ABZ和N 'HA的氧化，并建议ABZ的逐步氧化 转化为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.