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

摘要

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 N3 - 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 PPi. 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-PPi, 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.