Lamothe Maria, Chang Fu-Jung, Balashova Nataliya, Shirokov Roman, Beuve Annie
Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark, New Jersey 07103, USA.
Biochemistry. 2004 Mar 23;43(11):3039-48. doi: 10.1021/bi0360051.
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.
可溶性鸟苷酸环化酶（sGC）是一种由α亚基和β亚基组成的异二聚体酶，后者含有一氧化氮（NO）结合的血红素。当NO结合时，sGC的基础活性会增加数百倍。YC-1（一种苄基吲唑别构激活剂）也会增加sGC的活性。在NO存在的情况下，YC-1通过增强NO与血红素的亲和力来协同增加sGC的催化活性。YC-1与sGC的相互作用位点尚不清楚。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激活的传递。由于鸟苷酸环化酶（GCs）和腺苷酸环化酶（ACs）是同源的，我们利用AC的三维结构来指导诱变。对纯化突变体的生化分析表明，YC-1不仅通过增加NO亲和力来增加催化活性，还通过增加NO的效能来实现。单点突变使YC-1对NO亲和力和效能的影响分离，这意味着YC-1与sGC至少有两种类型的相互作用。一个结构模型预测，YC-1可能在一个与GTP结合位点伪对称且等同于AC中福斯可林位点的位点采取两种构象。
