Functional characterization of nitric oxide and YC-1 activation of soluble guanylyl cyclase: Structural implication for the YC-1 binding site?
Functional characterization of nitric oxide and YC-1 activation of soluble guanylyl cyclase: Structural implication for the YC-1 binding site?
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DOI：
10.1021/bi0360051
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发表时间：
2004-03-23
期刊：
影响因子：
2.9
通讯作者：
Beuve, A
中科院分区：
文献类型：
--
作者：
Lamothe, M;Chang, FJ;Beuve, A
可溶性鸟苷酸环化酶（sGC）是由α亚基和β亚基形成的异二聚体酶，后者含有一氧化氮（NO）结合的血红素。当NO结合时，sGC的基础活性增加数百倍。sGC活性也被YC-1（一种苄基吲唑变构激活剂）增加。在NO存在下，YC-1通过增强NO对血红素的亲和力来协同地增加sGC的催化活性。YC-1与sGC相互作用的位点是未知的。我们进行了突变分析，以确定结合位点，并确定哪些残基参与了NO和/或YC-1激活的传播。由于鸟苷酸环化酶（GC）和腺苷酸环化酶（AC）是同源的，我们使用AC的三维结构来指导诱变。纯化的突变体的生化分析表明，YC-1增加的催化活性，不仅通过增加NO的亲和力，但也通过增加NO的功效。YC-1对NO的亲和力和功效的影响被解离的单点突变，这意味着YC-1有，至少有两种类型的相互作用与sGC。一个结构模型预测，YC-1可能会采取两种配置在一个网站是伪对称的GTP结合位点和相当于毛喉素网站AC。
Soluble guanylyl cyclase (sGC) is a heterodimeric enzyme formed by an alpha subunit and a beta subunit, the latter containing the heme where nitric oxide (NO) binds. When NO binds, the basal activity of sGC is increased several hundred fold. sGC activity is also increased by YC-1, a benzylindazole allosteric activator. In the presence of NO, YC-1 synergistically increases the catalytic activity of sGC by enhancing the affinity of NO for the heme. The site of interaction of YC-1 with sGC is unknown. We conducted a mutational analysis to identify the binding site and to determine what residues were involved in the propagation of NO and/or YC-1 activation. Because guanylyl cyclases (GCs) and adenylyl cyclases (ACs) are homologous, we used the three-dimensional structure of AC to guide the mutagenesis. Biochemical analysis of purified mutants revealed that YC-1 increases the catalytic activity not only by increasing the NO affinity but also by increasing the efficacy of NO. Effects of YC-1 on NO affinity and efficacy were dissociated by single-point mutations implying that YC-1 has, at least, two types of interaction with sGC. A structural model predicts that YC-1 may adopt two configurations in one site that is pseudosymmetric with the GTP binding site and equivalent to the forskolin site in AC.