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

• Published in: The Journal of biological chemistry 2005-03, Vol.280 (12), p.11513-11519
• Main Authors: Chang, Fu-Jung, Lemme, Scott, Sun, Qian, Sunahara, Roger K., Beuve, Annie
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - metabolism; Adenosine Triphosphate - pharmacology; Allosteric Regulation; Binding Sites; Catalytic Domain; Guanosine 5'-O-(3-Thiotriphosphate) - pharmacology; Guanosine Triphosphate; Guanylate Cyclase - antagonists & inhibitors; Guanylate Cyclase - chemistry; Nitric Oxide - physiology; Nucleotides - metabolism; Nucleotides - pharmacology; Penicillamine - analogs & derivatives; Penicillamine - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M412203200

The mechanism of desensitization of the nitric oxide (NO) receptor (α1·β1 isoform of soluble guanylyl cyclase, sGC) is not known. Models of the structure of α1·β1, 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 Mg2+GTP (substrate) binding in α1·β1 into those present in AC fully reverts GC activity to AC activity. The wild-type form of α1·β1 (GC-wt) and the mutant form (AC-mut, α1R592Q·β1E473K,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, Mg2+GTPγS and Mg2+ATPγS 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 β1D477A, the pseudosymmetric equivalent to α1D529 (located in the substrate-binding site and involved in substrate binding and catalysis), or with the double mutations α1E525K,C594D, the pseudosymmetric equivalent to β1E473K,C541D. Taken together these data suggest that occupation of the second site by nucleotides may underlie part of the mechanism of desensitization of sGC.