title: Functional characterization of two nucleotide-binding sites in soluble guanylate cyclase.
abstract: Soluble guanylate cyclase is a heterodimeric hemoprotein composed of alpha- and beta-subunits with a homologous motif to the nucleotide-binding sites of adenylate cyclases. Homology modeling of guanylate cyclase, based on the crystal structure of adenylate cyclase, reveals a single GTP-binding site and a putative second site pseudosymmetric to the GTP-binding site. However, the role of this pseudosymmetric site has remained unclear. Using equilibrium dialysis, we identified two nucleotide-binding sites with high and low affinity for alpha,beta-methylene guanosine 5'-triphosphate (GMP-CPP). In contrast, 2'-dADP occupied both sites with equivalent affinities. Adenosine-5'-beta,gamma-imido triphosphate (AMP-PNP), which competitively inhibited the cyclase reaction, bound solely to the high affinity site, indicating the role of this site as the catalytic site. The function of the low affinity site was examined using allosteric activators YC-1 and BAY 41-2272. YC-1 significantly reduced the affinity of 2'-dADP, probably by competing for the same site as 2'-dADP. BAY 41-2272 totally inhibited the specific binding of one molecule of 2'-dADP as well as GMP-CPP. This suggests that the activators compete with these nucleotides for the low affinity site. Infrared and EPR analyses of the enzymic CO- and NO-hemes also supported the suggested role of the low affinity site as a target for the activators. Our results imply that the low affinity site is the pseudosymmetric site, which binds YC-1 or BAY 41-2272.
authors.affiliation: Department of Life Science, College of Science, Rikkyo University, Nishi-ikebukuro 3-34-1, Toshima-ku, Tokyo 171-8501.
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Yazawa
authors.affiliation: Department of Life Science, College of Science, Rikkyo University, Nishi-ikebukuro 3-34-1, Toshima-ku, Tokyo 171-8501.
authors.forename: Hidemi
authors.initials: H
authors.lastname: Tsuchiya
authors.affiliation: Division of Bioengineering, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hori
authors.affiliation: Department of Life Science, College of Science, Rikkyo University, Nishi-ikebukuro 3-34-1, Toshima-ku, Tokyo 171-8501. Electronic address: rmakino@rikkyo.ne.jp.
authors.forename: Ryu
authors.initials: R
authors.lastname: Makino
mesh_terms: D000244:Adenosine Diphosphate; D000249:Adenosine Monophosphate; D000262:Adenylyl Cyclases / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D020134:Catalytic Domain; D002417:Cattle; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives; D006160:Guanosine Triphosphate; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000378:metabolism; D008168:Lung / Q000201:enzymology; D009711:Nucleotides / Q000378:metabolism*; D012995:Solubility; D040681:Structural Homology, Protein; D013379:Substrate Specificity
chemical_list: D009711:Nucleotides; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D006157:Guanosine Monophosphate; D006160:Guanosine Triphosphate; D000262:Adenylyl Cyclases; D006162:Guanylate Cyclase