Functional Characterization of Two Nucleotide Binding Site in Soluble Guanylate Cyclase

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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.