Interactions of soluble guanylate cyclase with a P-site inhibitor: effects of gaseous heme ligands, azide, and allosteric activators on the binding of 2'-deoxy-3'-GMP.

Nitric oxide (NO) elicits a wide variety of physiological responses by binding to the heme in soluble guanylate cyclase (sGC) to stimulate cGMP production. Although nucleotides, such as ATP or GTP analogues, have been reported to regulate the signaling of NO binding from the heme site to the catalytic site, the other regulatory functions of nucleotides remain unexamined. Among the nucleotides tested, we found that 2'-d-3'-GMP acted as a potent noncompetitive inhibitor with respect to Mn-GTP, when the ferrous enzyme combined with NO, CO, or allosteric activator BAY 41-2272. 2'-d-3'-GMP also displayed nearly identical patterns of inhibition for the ferric enzyme, in which the binding of N(3)(-) or BAY 41-2272 significantly increased the inhibitory effects of the nucleotide. Equilibrium dialysis measurements using the CO-ligated enzyme in the presence of allosteric activators demonstrated that 2'-d-3'-GMP exclusively binds to the catalytic site of sGC. Furthermore, the affinity of 2'-d-3'-GMP for the enzyme was found to increase upon addition of foscarnet, an analogue of PP(i). These findings together with other kinetic results imply that 2'-d-3'-GMP acts as a P-site inhibitor probably by forming a dead-end complex, sGC-2'-d-3'-GMP-PP(i), in the catalytic reaction. The formation of the complex of the enzyme with 2'-d-3'-GMP does not seem to be associated with changes in the Fe-proximal His bond strength, because the CO coordination state or the redox potentials of the enzyme-heme complex are virtually unaffected.

Functional characterization of two nucleotide-binding sites in soluble guanylate cyclase.

Soluble guanylate cyclase is a heterodimeric hemoprotein composed of alpha- and beta-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 alpha,beta-methylene guanosine 5'-triphosphate (GMP-CPP). In contrast, 2'-dADP occupied both sites with equivalent affinities. Adenosine-5'-beta,gamma-imido triphosphate (AMP-PNP), which competitively inhibited the cyclase reaction, bound solely to the high affinity site, indicating the role of this site as the 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. This suggests 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.