Kosarikov D N, Lee J M, Uversky V N, Counts Gerber N
Department of Chemistry and Biochemistry, San Francisco State University, 1600 Holloway Avenue, San Francisco, CA 94132-4163, USA.
J Inorg Biochem. 2001 Dec 15;87(4):267-76. doi: 10.1016/s0162-0134(01)00387-7.
Soluble guanylate cyclase (sGC) is a receptor for endogenous and exogenous nitric oxide (NO) and is activated many fold upon its binding, making it a core enzyme in the nitric oxide signal transduction pathway. Much effort has been made to understand the link between binding of NO at the sGC heme and activation of the cyclase activity. We report here the first direct evidence for the role of conformational changes in transmitting the signal between the heme and cyclase domains. Using both circular dichroism (CD) and fluorescence spectroscopies, we have probed the effect that the sGC activators NO and 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl-indazole (YC-1) and the inhibitor 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ) have on the structure of the protein. Surprisingly, binding of either ODQ or YC-1 to NO-bound sGC cause virtually identical changes in the far-UV CD spectra of sGC, reflecting a perturbation in the secondary structure of the enzyme. This change is absent upon binding of NO, YC-1 or ODQ alone. Using this and previous data, we propose a working model for the mechanism of activation of sGC by NO and YC-1 and inhibition by ODQ.
可溶性鸟苷酸环化酶（sGC）是内源性和外源性一氧化氮（NO）的受体，在结合后会被激活许多倍，使其成为一氧化氮信号转导途径中的核心酶。人们为了解sGC血红素上NO的结合与环化酶活性激活之间的联系付出了很多努力。我们在此报告了构象变化在血红素和环化酶结构域之间传递信号中作用的首个直接证据。使用圆二色性（CD）光谱和荧光光谱，我们探究了sGC激活剂NO和3-(5'-羟甲基-2'-呋喃基)-1-苄基吲唑（YC-1）以及抑制剂1H-[1,2,4]-恶二唑并-[4,3-a]-喹喔啉-1-酮（ODQ）对蛋白质结构的影响。令人惊讶的是，ODQ或YC-1与结合了NO的sGC结合，会在sGC的远紫外CD光谱中引起几乎相同的变化，反映出该酶二级结构的扰动。单独结合NO、YC-1或ODQ时不会出现这种变化。利用这些以及先前的数据，我们提出了一个关于NO和YC-1激活sGC以及ODQ抑制sGC机制的工作模型。
