Nitric Oxide-dependent Allosteric Inhibitory Role of a Second Nucleotide Binding Site in Soluble Guanylyl Cyclase

Abstract

The mechanism of desensitization of the nitric oxide (NO) receptor (␣ 1 ⅐␤ 1 isoform of soluble guanylyl cyclase, sGC) is not known. Models of the structure of ␣ 1 ⅐␤ 1 , based on the x-ray crystal structure of adenylyl cyclase (AC) suggest the existence of a nucleotide-like binding site, in addition to the putative catalytic site. We have previously reported that mutating residues that coordinate Mg 2؉ GTP (substrate) binding in ␣ 1 ⅐␤ 1 into those present in AC fully reverts GC activity to AC activity. The wild-type form of ␣ 1 ⅐␤ 1 (GC-wt) and the mutant form (AC-mut, ␣ 1 R592Q⅐␤ 1 E473K,C541D) were purified, and their sensitivities to various nucleotides were assessed. In using the AC-mut as well as other mutants that coordinate purine binding, we were able to distinguish allosteric inhibitory effects of guanine nucleotides from competitively inhibitory effects on catalytic activity. Here we report that several nucleotide analogs drastically alter sGC and AC-mut activity by acting at a second nucleotide site, likely pseudosymmetric to the catalytic site. In particular, Mg 2؉ GTP␥S and Mg 2؉ ATP␥S inhibited cyclase activity through a mixed, non-competitive mechanism that was only observable under NO stimulation and not under basal conditions. The non-competitive pattern of inhibition was not present in mutants carrying the substitution ␤ 1 D477A, the pseudosymmetric equivalent to ␣ 1 D529 (located in the substrate-binding site and involved in substrate binding and catalysis), or with the double mutations ␣ 1 E525K,C594D, the pseudosymmetric equivalent to ␤ 1 E473K,C541D. Taken together these data suggest that occupation of the second site by nucleotides may underlie part of the mechanism of desensitization of sGC.

Tonic and acute nitric oxide signaling through soluble guanylate cyclase is mediated by nonheme nitric oxide, ATP, and GTP

“…A recent modeling and mutational study suggests that both ATP and GTP bind to a pseudosubstrate site in the catalytic domains (22); however, it remains a possibility that ATP and͞or GTP might bind elsewhere on the enzyme.…”

“…Recent data also suggest that full activation of sGC by NO at the heme requires high levels of the cyclase reaction products [cGMP and͞or pyrophosphate (PP i )] before NO binding (21). Furthermore, ATP at physiological concentrations inhibits sGC activity by as much as 50% (22,23). Clearly, NO and nucleotides affect sGC function in ways not previously appreciated.…”

Functional Characterization of Two Nucleotide-binding Sites in Soluble Guanylate Cyclase

“…The pseudosymmetric site contains a Gly residue at the position corresponding to ␣Asp 485 within the GTP-binding site (27,29). Therefore, it is feasible that the replacement of the Asp residue by a Gly results in destabilization of the interaction between the phosphate moieties of nucleotide.…”

“…Based on the homology modeling of sGC, the GTP-binding site contains two Asp residues (␣Asp 485 and ␣Asp 529 ) that are critical for metal coordination and hence for binding the phosphate moiety of the nucleotide (27,29). The pseudosymmetric site contains a Gly residue at the position corresponding to ␣Asp 485 within the GTP-binding site (27,29).…”

“…By analogy with the forskolin-binding site of AC, one might predict that the pseudosymmetric site constitutes the binding site for YC-1 (3-(5Ј-hydroxymethyl-3Ј-furyl)-1-benzylindazol), which is known to sensitize sGC to activation by NO and CO (28). Furthermore, Chang et al (29) has shown that ATP analogue ATP␥S alters the cyclase activity presumably by acting at the pseudosymmetric site.…”