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首页> 外文期刊>Biochemistry >The P450_cam G248E Mutant Covalently Binds Its Prosthetic Heme Group
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The P450_cam G248E Mutant Covalently Binds Its Prosthetic Heme Group

机译：P450_cam G248E突变体共价结合了其人工血红素基团

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Previous studies on mammalian peroxidases and cytochrome P450 family 4 enzymes have shown that a carboxylic group positioned close to a methyl group of the prosthetic heme is required for the formation of a covalent link between a protein carboxylic acid side chain and the heme.To determine whether there are additional requirements for covalent bond formation in the P450 enzymes,a glutamic acid or an aspartic acid has been introduced into P450_cam close to the heme 5-methyl group.Spectroscopic and kinetic studies of the resulting G248E and G248D mutants suggest that the carboxylate group coordinates with the heme iron atom,as reported for a comparable P450_BM3 mutant [Girvan,H.M.,Marshall,K.R.,Lawson,R.J.,Leys,D.,Joyce,M.G.,Clarkson,J.,Smith,W.E.,Cheesman,M.R.,and Munro,A.W.(2004)J.Biol.Chem.279,23274-23286].The two P450_cam mutants have low catalytic activity,but in contrast to the P450_BM3 mutant,incubation of the G248E(but not G248D)mutant with camphor,putidaredoxin,putidaredoxin reductase,and NADH results in partial covalent binding of the heme to the protein.No covalent attachment is observed in the absence of camphor or any of the other reaction components.Pronase digestion of the G248E P450_cam mutant after covalent attachment of the heme releases 5-hydroxyheme,establishing that the heme is covalently attached through its 5-methyl group as predicted by in silico modeling.The results establish that a properly positioned carboxyl group is the sole requirement for autocatalytic formation of a heme-protein link in P450 enzymes,but also show that efficient covalent binding requires placement of the carboxyl close to the methyl but in a manner that prevents strong coordination to the iron atom.
机译：以前对哺乳动物过氧化物酶和细胞色素P450家族4酶的研究表明，在蛋白质羧酸侧链与血红素之间形成共价键时，需要在人造血红素的甲基附近设置一个羧基。在P450酶中还需要形成共价键，谷氨酸或天冬氨酸已被引入到P450_cam中靠近血红素5-甲基的位置。所得G248E和G248D突变体的光谱和动力学研究表明，羧酸酯基团如可比较的P450_BM3突变体[Girvan，HM，Marshall，KR，Lawson，RJ，Leys，D.，Joyce，MG，Clarkson，J.，Smith，WE，Cheesman，MR，和Munro，AW（2004）J.Biol.Chem.279，23274-23286]。这两个P450_cam突变体具有较低的催化活性，但是与P450_BM3突变体相反，将G248E（而不是G248D）突变体与樟脑，扑热还原蛋白一起孵育普达氧还蛋白酶和NADH导致血红素与蛋白质部分共价结合。在没有樟脑或任何其他反应组分的情况下，未观察到共价结合。血红素共价结合后G248E P450_cam突变体的磷酸酶消化释放了5-羟基血红素，建立了在计算机模拟中预测的血红素通过其5-甲基共价连接的结果。结果表明，正确定位的羧基是在P450酶中自动催化形成血红素-蛋白质连接的唯一条件，而且结果表明，有效的共价结合需要将羧基放置在靠近甲基的位置，但是要防止与铁原子的强配位。

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