title: 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 (alpha1.beta1 isoform of soluble guanylyl cyclase, sGC) is not known. Models of the structure of alpha1.beta1, 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 alpha1.beta1 into those present in AC fully reverts GC activity to AC activity. The wild-type form of alpha1.beta1 (GC-wt) and the mutant form (AC-mut, alpha1R592Q.beta1E473K,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 gamma S and Mg(2+)ATP gamma 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 beta1D477A, the pseudosymmetric equivalent to alpha1D529 (located in the substrate-binding site and involved in substrate binding and catalysis), or with the double mutations alpha1E525K,C594D, the pseudosymmetric equivalent to beta1E473K,C541D. Taken together these data suggest that occupation of the second site by nucleotides may underlie part of the mechanism of desensitization of sGC.
authors.affiliation: Department of Pharmacology and Physiology, New Jersey Medical School-UMDNJ, Newark, New Jersey 07103, USA.
authors.forename: Fu-Jung
authors.initials: FJ
authors.lastname: Chang
authors.forename: Scott
authors.initials: S
authors.lastname: Lemme
authors.forename: Qian
authors.initials: Q
authors.lastname: Sun
authors.forename: Roger K
authors.initials: RK
authors.lastname: Sunahara
authors.forename: Annie
authors.initials: A
authors.lastname: Beuve
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000494:Allosteric Regulation; D001665:Binding Sites; D020134:Catalytic Domain; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D009569:Nitric Oxide / Q000502:physiology*; D009711:Nucleotides / Q000378:metabolism / Q000494:pharmacology*; D010396:Penicillamine / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D009711:Nucleotides; C110881:S-nitro-N-acetylpenicillamine; C036852:magnesium GTP; D009569:Nitric Oxide; C022571:adenosine 5'-O-(3-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D006162:Guanylate Cyclase; D010396:Penicillamine