Allosteric inhibition by adenine nucleotides is mediated by direct interaction with sGC. (A and B) ATP and 8N₃ATP inhibit purified human sGC stimulated by 100 μM SNP with comparable potencies. Reaction rates are expressed as fractional response, described in Fig. 1. ▪, ATP; ▴, 8N₃ATP. Reaction rates presented in sigmoidal plots (A) were subjected to double reciprocal analysis (B). (C)8N₃ATP specifically interacts with a site on purified human sGC comprised of residues contributed by α₁- and β₁-subunits. Purified enzyme (1 μg) was incubated in the absence (pos, positive control) or presence of 0.1, 1, or 10 mM ATP for 10 min, followed by addition of 50 μM 8-azido [γ³²P]ATP. Reactions were incubated for 10 min and, unless otherwise indicated (neg, negative control), irradiated at 254 nm by 4.4 milliwatts/cm² for 2 min. Photoaffinity labeling was detected by autoradiography (Upper) and protein with GelCode blue (Lower).

Inhibition of sGC by ATP is mediated by an allosteric purine nucleotide binding site. (A) Binding of 8N₃ATP to human purified sGC is antagonized by purine nucleotides with a rank order potency of ATP > GTP. Enzyme (1 μg) was preincubated with 1 mM ATP or GTP and photoaffinity-labeled with 50 μM 8-azido [γ³²P]ATP, as outlined in Fig. 2. Relative incorporation of radioactivity into sGC in the presence of unlabeled nucleotides is expressed as the percentage of displacement compared to incubations that omitted those nucleotides. (B–E) GTP antagonizes inhibition of sGC by ATP by productively interacting with the allosteric nucleotide binding site. (B) GTP shifted the concentration dependence of ATP inhibition to the right, reflecting the ability of GTP to compete with ATP for binding to the nucleotide regulatory site. Enzyme activity stimulated by 100 μM SNP was quantified by using 50 μM(▪) or 1 mM(▴) GTP. Reaction rates are expressed as fractional response, described in Fig. 1. Reaction rates of purified sGC stimulated by SNP with 50 μM GTP or 1 mM GTP were 619 ± 76 and 3,205.2 ± 1,296 nmol cGMP min^–1·mg^–1 of protein, respectively. Reaction rates presented in sigmoidal plots (B) were subjected to double reciprocal analysis (C). The K_i of ATP, estimated by nonlinear regression analyses of sigmoidal plots (n = 3), was significantly (P < 0.05) greater when higher concentrations of GTP were used (Inset). (D) Purified human sGC activity stimulated by 100 μM SNP was quantified by using a range of [GTP] in the presence (▴) or absence (▪) of 1 mM ATP. Indeed, [GTP] >1 mM directly inhibit, and attenuate the ability of ATP to inhibit, sGC. The concentration dependence of GTP to attenuate inhibition of sGC by ATP, reflecting productive interaction of GTP with the allosteric nucleotide regulatory site, is illustrated by plotting relative enzyme activities (activity in the absence of ATP/activity in the presence of ATP; V/V_ATP) (E). In A and E, results represent the means ± SEM of three experiments. *, P < 0.05 compared to 0.1 mM GTP.