Chen P F, Wu K K
Vascular Biology Research Center and Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center, Houston, Texas 77225, USA.
J Biol Chem. 2000 Apr 28;275(17):13155-63. doi: 10.1074/jbc.275.17.13155.
It has been postulated that a segment (residues 594-645) inserted in the FMN subdomain of human endothelial nitric-oxide synthase (eNOS) plays a crucial role in controlling Ca(2+)-dependent CaM binding for eNOS activity. To investigate its functions, we expressed human eNOS in a baculovirus system with deletion of a 45-residue segment from this region (residues 594-606 and 614-645, designated as Delta45eNOS), and characterized the purified mutant enzyme. In contrast with wild-type eNOS, Delta45eNOS exhibited characteristics resembling inducible NOS (iNOS). It contained an endogenously bound CaM, which was essential in folding and stabilizing this mutant enzyme, and retained 60% of L-citrulline formation in 5 mM EGTA. We also produced four N-terminally truncated reductase domains with or without the 45-residue segment, and either including or excluding the CaM-binding sequence. Basal cytochrome c reductase activity of reductase domains without the 45-residue segment was up to 20 fold greater than that of corresponding insert-containing domains, and higher than CaM-stimulated activity of the wild-type enzyme. A series of mutants with smaller fragment deletion in this region such as Delta594-604, Delta605-612, Delta613-625, Delta626-634, Delta632-639, and Delta640-645 mutants were further characterized. The crude lysate of mutants Delta613-625 and Delta632-639 did not show activity in the presence of Ca(2+)/CaM, while other four mutants had activity comparable to that of WTeNOS. The purified Delta594-604 and Delta605-612 proteins had a 3-5-fold higher affinity for Ca(2+)/CaM, but their L-citrulline forming activity was still 80% dependent upon the addition of Ca(2+)/CaM. Both mutants exhibited a low level of the cytochrome c and ferricyanide reductase activities, which either did not respond to (Delta594-604) or slightly enhanced by (Delta605-612) the exogenous CaM. In contrast, activities of Delta626-634 and Delta640-645 like those of WTeNOS were largely Ca(2+)/CaM-dependent. Thus, our findings indicate that the N-terminal half of the 594-645 segment containing residues 594-612 plays a significant role in regulating Ca(2+)/CaM binding.
据推测，插入人内皮型一氧化氮合酶（eNOS）的黄素单核苷酸（FMN）亚结构域中的一段序列（第594 - 645位氨基酸残基）在控制eNOS活性的钙（Ca2+）依赖性钙调蛋白（CaM）结合中起关键作用。为了研究其功能，我们在杆状病毒系统中表达了缺失该区域45个氨基酸残基片段（第594 - 606位和614 - 645位氨基酸残基，命名为Delta45eNOS）的人eNOS，并对纯化的突变酶进行了表征。与野生型eNOS不同，Delta45eNOS表现出类似于诱导型一氧化氮合酶（iNOS）的特征。它含有内源性结合的CaM，这对该突变酶的折叠和稳定至关重要，并且在5 mM乙二醇双（2-氨基乙基醚）四乙酸（EGTA）中保留了60%的L-瓜氨酸生成能力。我们还制备了四个N端截短的还原酶结构域，分别带有或不带有45个氨基酸残基的片段，并且分别包含或不包含CaM结合序列。没有45个氨基酸残基片段的还原酶结构域的基础细胞色素c还原酶活性比相应含插入片段的结构域高20倍，且高于野生型酶的CaM刺激活性。对该区域中片段缺失更小的一系列突变体，如Delta594 - 604、Delta605 - 612、Delta613 - 625、Delta626 - 634、Delta632 - 639和Delta640 - 645突变体进行了进一步表征。Delta613 - 625和Delta632 - 639突变体的粗裂解物在存在Ca2+/CaM时没有活性，而其他四个突变体具有与野生型eNOS相当的活性。纯化的Delta594 - 604和Delta605 - 612蛋白对Ca2+/CaM的亲和力高3 - 5倍，但其L-瓜氨酸生成活性仍有80%依赖于添加Ca2+/CaM。这两个突变体均表现出低水平的细胞色素c和铁氰化物还原酶活性，其中Delta594 - 604对（外源性）CaM无反应，Delta605 - 612则略有增强。相反，Delta626 - 634和Delta640 - 645的活性与野生型eNOS一样，在很大程度上依赖于Ca2+/CaM。因此，我们的研究结果表明，包含第594 - 612位氨基酸残基的594 - 645片段的N端一半在调节Ca2+/CaM结合中起重要作用。
