Identification of Tyr504 as an alternative tyrosyl radical site in human prostaglandin H synthase-2.
Identification of Tyr504 as an alternative tyrosyl radical site in human prostaglandin H synthase-2.
复制标题
鉴定 Tyr504 作为人前列腺素 H 合酶 2 中的替代酪氨酰基位点。
DOI：
10.1021/bi035717o
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Tsai,Ah-Lim
中科院分区：
文献类型：
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作者：
Rogge,CorinaE;Liu,Wen;Wu,Gang;Wang,Lee-Ho;Kulmacz,RichardJ;Tsai,Ah-Lim
氢过氧化物诱导前列腺素H合成酶（PGHS） Tyr385上酪氨酸基自由基的形成。Tyr385自由基启动花生四烯酸的氢提取，从而机械地连接过氧化物酶和环氧合酶的活性。在这两种PGHS同工异构体中，酪氨酸自由基经历了从宽重态到宽单重态的时间依赖性转变；环加氧酶抑制剂预处理导致第三类信号，窄单线态[Tsai， a .- l .；库尔马兹，r.j.（2000）。脂质医学。62,231−254]。这些转变被解释为Tyr385环旋转的结果，但也可能是由于Tyr385自由基迁移到另一个酪氨酸残基。通过对PGHS晶体结构的pathway分析，确定了4个预测电子耦合良好的酪氨酸残基：残基148、348、404和504（羊PGHS-1编号）。我们表达了含有单个Tyr→Phe突变的重组PGHS-2蛋白，一个包含所有四个酪氨酸突变的四重突变体，以及一个包含Y385F突变的五重突变体。所有突变体结合血红素并表现出明显的过氧化物酶活性，除了五重突变体外，所有突变体都保留环加氧酶活性，这表明活性位点都没有受到明显干扰。Y148F、Y348F和Y404F突变体与eooh反应后，产生与天然PGHS-2相似的宽单线EPR信号。然而，Y504F和四重突变体与过氧化物反应产生持久的宽双链，而五重突变体是EPR沉默的。尼美舒利预处理Y504F和四重突变体导致异常少量的宽双线信号，没有形成窄单线信号。因此，Tyr504上替代酪氨酸自由基的形成可能解释了天然PGHS-2从宽双线态到宽单线态的转变，以及PGHS-2与环加氧酶抑制剂配合物形成窄单线态的原因。
Hydroperoxides induce formation of a tyrosyl radical on Tyr385 in prostaglandin H synthase (PGHS). The Tyr385 radical initiates hydrogen abstraction from arachidonic acid, thereby mechanistically connecting the peroxidase and cyclooxygenase activities. In both PGHS isoforms the tyrosyl radical undergoes a time-dependent transition from a wide doublet to a wide singlet species; pretreatment with cyclooxygenase inhibitors results in a third type of signal, a narrow singlet [Tsai, A.-L.; Kulmacz, R. J. (2000)Prost. Lipid Med. 62, 231−254]. These transitions have been interpreted as resulting from Tyr385 ring rotation, but could also be due to radical migration from Tyr385 to another tyrosine residue. PATHWAYS analysis of PGHS crystal structures identified four tyrosine residues with favorable predicted electronic coupling: residues 148, 348, 404, and 504 (ovine PGHS-1 numbering). We expressed recombinant PGHS-2 proteins containing single Tyr → Phe mutations at the target residues, a quadruple mutant with all four tyrosines mutated, and a quintuple mutant, which also contains a Y385F mutation. All mutants bind heme and display appreciable peroxidase activity, and with the exception of the quintuple mutant, all retain cyclooxygenase activity, indicating that neither of the active sites is significantly perturbed. Reaction of the Y148F, Y348F, and Y404F mutants with EtOOH generates a wide singlet EPR signal similar to that of native PGHS-2. However, reaction of the Y504F and the quadruple mutants with peroxide yields persistent wide doublets, and the quintuple mutant is EPR silent. Nimesulide pretreatment of Y504F and the quadruple mutant results in an abnormally small amount of wide doublet signal, with no narrow singlet being formed. Therefore, the formation of an alternative tyrosine radical on Tyr504 probably accounts for the transition from a wide doublet to a wide singlet in native PGHS-2 and for formation of a narrow singlet in complexes of PGHS-2 with cyclooxygenase inhibitors.