STRUCTURAL-SIGNIFICANCE AND FUNCTIONAL-SIGNIFICANCE OF CYSTEINE RESIDUES OF GLUTATHIONE-INDEPENDENT PROSTAGLANDIN-D SYNTHASE - IDENTIFICATION OF CYS(65) AS AN ESSENTIAL THIOL
STRUCTURAL-SIGNIFICANCE AND FUNCTIONAL-SIGNIFICANCE OF CYSTEINE RESIDUES OF GLUTATHIONE-INDEPENDENT PROSTAGLANDIN-D SYNTHASE - IDENTIFICATION OF CYS(65) AS AN ESSENTIAL THIOL
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DOI：
10.1074/jbc.270.3.1422
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发表时间：
1995-01-20
影响因子：
4.8
通讯作者：
HAYAISHI, O
中科院分区：
文献类型：
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作者：
URADE, Y;TANAKA, T;HAYAISHI, O
大鼠脑中不依赖谷胱甘肽的前列腺素D合酶由189个氨基酸残基组成，它催化前列腺素H - 2异构化为前列腺素D - 2，后者是一种内源性促睡眠物质。这种酶是脂质运载蛋白超家族成员中唯一的一种酶，该超家族由各种分泌性亲脂性配体载体蛋白组成，并且最近被鉴定为β - 痕迹蛋白，是人类脑脊液的主要成分。我们在大肠杆菌中表达了有活性的酶，然后系统地将缺失1 - 29个氨基酸的酶在65、89和186位的所有半胱氨酸残基用丙氨酸或丝氨酸替代。通过葡聚糖凝胶G - 50和S - 琼脂糖凝胶层析，将亲本酶和突变酶纯化至表观均一性，回收率约为30%，所有酶都显示出相同的洗脱曲线。无论是否突变，纯化后的酶都显示出与高度有序的β - 结构所呈现的几乎相同的圆二色性光谱特征。含有半胱氨酸65的重组酶在有巯基化合物存在时，其活性与从大鼠脑中纯化的酶相当（约3 μmol/min/mg蛋白质），但在无巯基化合物时则无活性。然而，所有不含半胱氨酸65的单突变体、双突变体和三突变体都失去了酶活性。纯化的缺失1 - 29且丙氨酸在89和186位的酶在半胱氨酸65位与谷胱甘肽结合时可逆地失活，并且被N - 乙基马来酰亚胺按化学计量进行化学修饰时不可逆地失活。这些结果表明半胱氨酸65是该酶的一个必需巯基，并且内在和外在的巯基对于该酶催化前列腺素H - 2的9,11 - 内过氧化物进行非氧化重排以产生前列腺素D - 2都是必需的。
Glutathione-independent prostaglandin D synthase in rat brain is composed of 189 amino acid residues and catalyzes the isomerization of prostaglandin H-2,to prostaglandin D-2, an endogenous sleep-promoting substance. This enzyme is the only enzyme among members of the lipocalin superfamily composed of various secretory lipophilic ligand-carrier proteins and is recently identified to be a beta-trace protein, a major constituent of human cerebrospinal fluid. We expressed the active enzyme in Escherichia coli and then systematically substituted all cysteine residues of the Delta 1-29 enzyme at positions of 65, 89, and 186 with alanine or serine. The parent and mutant enzymes were purified to apparent homogeneity with a recovery of similar to 30% by chromatography with Sephadex G-50 and S-Sepharose, by which all the enzymes showed identical elution profiles. The purified enzymes, irrespective of the mutation, showed almost the same circular dichroism spectral characteristics as displayed by a highly ordered beta-structure. The recombinant enzymes containing Cys(65) showed the activity comparable with that of the enzyme purified hom rat brain (similar to 3 mu mol/min/mg of protein) in the presence, but not in the absence, of sulfhydryl compounds. However, all of the single, double, and triple mutants without Cys(65) lost the enzyme activity. The purified Delta 1-29 Ala(89,186) enzyme was inactivated reversibly by conjugation with glutathione at Cys(65) and irreversibly by the stoichiometric chemical modification with N-ethylmaleimide. These results indicate that Cys(65) is an essential thiol of the enzyme and that both the intrinsic and extrinsic sulfhydryl groups are necessary for nonoxidative rearrangement of 9,11-endoperoxide of prostaglandin H-2 to produce prostaglandin D-2 catalyzed by the enzyme.