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

Abstract

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.

Mechanistic Insights into the Activation of Soluble Guanylate Cyclase by Carbon Monoxide: A Multistep Mechanism Proposed for the BAY 41-2272 Induced Formation of 5-Coordinate CO–Heme

“…The 1964 and 1973 cm –1 modes had been assigned to a 5- and a 6-coordinate CO–heme, respectively, and their peak positions were essentially the same as those of the BAY-treated sGC–CO, except for slightly higher frequency shift by 2 cm –1 in the YC-1 bound 6-coordinate CO-heme. Elevating temperature intensified the 1964 and 1973 cm –1 modes, probably by enhancing the affinity of YC-1 . The binding of 2′-d-3′-GMP to the YC-1 bound form of sGC–CO intensified the 1964 and 1973 cm –1 bands with a concomitant loss of the 1987 cm –1 band (Figure S2 of the Supporting Information).…”

“…Elevating temperature intensified the 1964 and 1973 cm −1 modes, probably by enhancing the affinity of YC-1. 25 The binding of 2′-d-3′-GMP to the YC-1 bound form of sGC−CO intensified the 1964 and 1973 cm −1 bands with a concomitant loss of the 1987 cm −1 band (Figure S2 of the Supporting Information). The peak height of the 1964 cm −1 band was prominently intensified by the further addition of foscarnet (Figure S3 of the Supporting Information), in which the relative band area increased upon lowering temperature (inset in Figure S3A of the Supporting Information).…”

“…The latter 1987 cm −1 band can be virtually ignored due to its small contribution at all temperatures investigated. The 1964 and 1971 cm −1 bands comprised in the overlapping νC−O band were assigned to the νC−O mode of 5-and 6-coordinate CO−heme, respectively, 19,25 both of which were in a conformational state with a single Fe-CO orientation. The νC−O peak position of the 5coordinate CO−heme is located in the lower wavelength region than that of the 6-coordinate CO−heme, as reported in the previous heme-model study.…”

“…Our previous kinetic study indicated that 2′-d-3′-GMP acts as a potent purine-site (P-site) inhibitor by binding to the catalytic site of the BAY–sGC–CO complex together with pyrophosphate (PPi). While the P-site inhibitor complex [BAY–(sGC–CO)–2′-d-3′-GMP–PPi complex] could not be isolated as stable species, it was stabilized when foscarnet was used as an analogue of PPi in the presence of Mn 2+ . The resultant P-site inhibitor complex [BAY–(sGC–CO)–2′-d-3′-GMP–foscarnet] which is schematically illustrated below, is substantially equivalent to the BAY–(sGC–CO)–GTP complex, in respect that 2′-d-3′-GMP and foscarnet occupied the nucleotide and phosphate recognition sites, respectively.…”

A new small molecule inhibitor of soluble guanylate cyclase

“…We have identified a new type of small molecule inhibitors of sGC, which are thought to be the first class to act through allosteric regulation of the enzyme catalytic domain, rather than oxidation of the haem or through the purine p-site. 36 It is possible that the inhibitors presented bind to the catalytic domain of sGC inducing a conformational change, or ‘locking’ the enzyme in a basal conformation, that is not favourable to activation by NO or GTP binding. The inhibitor also inhibits particulate GC but not the related adenylate cyclase.…”