Functional Characterization of Two Nucleotide Binding Site in Soluble Guanylate Cyclase

可溶性鸟苷酸环化酶中两个核苷酸结合位点的功能表征

• 批准号: 15570124
• 负责人: MAKINO Ryu
• 金额: $ 2.11万
• 依托单位: Rikkyo University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15570124/
• 关键词: guanylate cyclase; nitric oxide; YC-1; BAY 41-2272; nucleotide; carbon monoxide; 一酸化窒素(NO); 一酸化炭素

Soluble guanylate cyclase is a heterodimeric hemoprotein composed of α- and β-subunits with a homologous motif to the nucleotide-binding sites of adenylate cyclases. Homology modeling of guanylate cyclase, based on the crystal structure of adenylate cyclase, reveals a single GTP-binding site and a putative second site pseudosymmetric to the GTP-binding site. However, the role of this pseudosymmetric site has remained unclear. Using equilibrium dialysis, we identified two nucleotide binding sites with high and low affinity for GMP-CPP (α,β-methylene GTP). In contrast to GMP-CPP binding, 2'-dADP occupied both sites with equivalent affinities. AMP-PNP (β,γ-imido ATP) which competitively inhibited the cyclase reaction, bound solely to the high-affinity site, indicating the role of this site as catalytic site. The function of the low-affinity site was examined using allosteric activators, YC-1 and BAY 41-2272. YC-1 significantly reduced the affinity of 2'-dADP, probably by competing for the same site as 2'-dADP. BAY 41-2272 totally inhibited the specific binding of one molecule of 2'-dADP as well as GMP-CPP. These suggest that the activators compete with these nucleotides for the low-affinity site. Infrared and EPR analyses of the enzymic CO- and NO-hemes also supported the suggested role of the low-affinity site as a target for the activators. Our results imply that the low-affinity site is the pseudosymmetric site, which binds YC-1 or BAY 41-2272.

可溶性鸟苷酸环化酶是一种异源二聚体血红素蛋白，由α-和β-亚基组成，具有与腺苷酸环化酶的核苷酸结合位点同源的基序。基于腺苷酸环化酶的晶体结构，鸟苷酸环化酶的同源性建模揭示了一个单一的GTP结合位点和一个假定的第二个与GTP结合位点假对称的位点。然而，这个假对称位点的作用仍然不清楚。利用平衡透析法，我们鉴定了两个对GMP CPP（α，β-methylene GTP）具有高亲和力和低亲和力的核苷酸结合位点。与GMP-CPP结合相反，2 '-dADP以相等的亲和力占据两个位点。竞争性抑制环化酶反应的AMP-PNP（β，γ-imido ATP）仅与高亲和力位点结合，表明该位点为催化位点。使用别构激活剂YC-1和BAY 41-2272检查低亲和力位点的功能。YC-1显著降低了2 '-dADP的亲和力，可能是通过与2'-dADP竞争相同的位点。BAY 41-2272完全抑制一分子2 ′-dADP和GMP-CPP的特异性结合。这表明激活剂与这些核苷酸竞争低亲和力位点。红外和EPR分析的酶CO-和NO-血红素也支持建议的低亲和力网站作为目标的激活剂的作用。我们的结果表明，低亲和力位点是假对称位点，它结合YC-1或BAY 41-2272。