Ability of tetrahydrobiopterin analogues to support catalysis by inducible nitric oxide synthase: formation of a pterin radical is required for enzyme activity.
Ability of tetrahydrobiopterin analogues to support catalysis by inducible nitric oxide synthase: formation of a pterin radical is required for enzyme activity.
复制标题
四氢生物蝶呤类似物支持诱导型一氧化氮合酶催化的能力：酶活性需要形成蝶呤自由基。
DOI：
10.1021/bi035491p
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发表时间：
2003
期刊：
影响因子：
2.9
通讯作者：
Marletta,MichaelA
中科院分区：
文献类型：
--
作者：
Hurshman,AmyR;Krebs,Carsten;Edmondson,DaleE;Marletta,MichaelA
用四氢生物蝶呤（H4B）或四氢生物蝶呤类似物（5-甲基-H4B和4-氨基-H4B）重组无pterin诱导型一氧化氮合酶（iNOS），并检测结合的5-甲基-H4B和4-氨基-H4B支持全长iNOS （FLiNOS）或分离血红素结构域（HDiNOS）催化的能力。在与HDiNOS的单次转换中，5-甲基- h4b形成非常稳定的自由基，5-甲基-H3B•，在精氨酸反应中积累到HDiNOS浓度的60%，衰变速度比H3B•慢约400倍（0.0003 vs 0.12 s-1）。在NHA反应中观察到的自由基（5-甲基-H3B•或H3B•）的量很小（< HDiNOS的3%）。5-甲基-H4B饱和FLiNOS和HDiNOS的活性与H4B结合时相似：精氨酸被羟基化成NHA， NHA被氧化成瓜氨酸和•NO。4-氨基- h4b或无pterin的HDiNOS均未观察到pterin自由基。4-氨基- h4b结合的FLiNOS和HDiNOS的催化活性与不含pterin的iNOS相似：精氨酸的羟基化非常不利（低于h4b结合的iNOS的2%），NHA被氧化成氨基酸产物（瓜氨酸和氰鸟氨酸）和NO的混合物，而不是•NO。这些结果表明，结合的蝶呤辅助因子经历了单电子氧化（形成蝶呤自由基），这对其支持正常NOS转换的能力至关重要。虽然H4B的结合也稳定了NOS的结构和活性位点，但pterin辅因子在NOS中最关键的作用似乎是电子转移。
Pterin-free inducible nitric oxide synthase (iNOS) was reconstituted with tetrahydrobiopterin (H4B) or tetrahydrobiopterin analogues (5-methyl-H4B and 4-amino-H4B), and the ability of bound 5-methyl-H4B and 4-amino-H4B to support catalysis by either full-length iNOS (FLiNOS) or the isolated heme domain (HDiNOS) was examined. In a single turnover with HDiNOS, 5-methyl-H4B forms a very stable radical, 5-methyl-H3B•, that accumulates in the arginine reaction to ∼60% of the HDiNOS concentration and decays ∼400-fold more slowly than H3B•(0.0003 vs 0.12 s-1). The amount of radical (5-methyl-H3B•or H3B•) observed in the NHA reaction is very small (<3% of HDiNOS). The activity of 5-methyl-H4B-saturated FLiNOS and HDiNOS is similar to that when H4B is bound: arginine is hydroxylated to NHA, and NHA is oxidized exclusively to citrulline and•NO. A pterin radical was not observed with 4-amino-H4B- or pterin-free HDiNOS with either substrate. The catalytic activity of 4-amino-H4B-bound FLiNOS and HDiNOS resembles that of pterin-free iNOS: the hydroxylation of arginine is very unfavorable (<2% that of H4B-bound iNOS), and NHA is oxidized to a mixture of amino acid products (citrulline and cyanoornithine) and NO-rather than•NO. These results demonstrate that the bound pterin cofactor undergoes a one-electron oxidation (to form a pterin radical), which is essential to its ability to support normal NOS turnover. Although binding of H4B also stabilizes the NOS structure and active site, the most critical role of the pterin cofactor in NOS appears to be in electron transfer.