title: The Influence of Nitric Oxide on Soluble Guanylate Cyclase Regulation by Nucleotides: ROLE OF THE PSEUDOSYMMETRIC SITE.
abstract: Activation of soluble guanylate cyclase (sGC) by the signaling molecule nitric oxide (NO) leads to formation of the second messenger cGMP, which mediates numerous physiological processes. NO activates sGC by binding to the ferrous heme cofactor; the relative amount of NO with respect to sGC heme affects the enzyme activity. ATP can also influence the activity by binding to an allosteric site, most likely the pseudosymmetric site located in the catalytic domain. Here, the role of the pseudosymmetric site on nucleotide regulation was investigated by point mutations at this site. ATP inhibition kinetics of wild type and a pseudosymmetric site (α1-C594A/β1-D477A) variant of sGC was determined at various levels of NO. Results obtained show that in the presence of less than 1 eq of NO, there appears to be less than complete activation and little change in the nucleotide binding parameters. The most dramatic effects are observed for the addition of excess NO, which results in an increase in the affinity of GTP at the catalytic site and full activation of sGC. The pseudosymmetric site mutation only affected nucleotide affinities in the presence of excess NO; there was a decrease in the affinity for ATP in both the allosteric and catalytic sites. These observations led to a new kinetic model for sGC activity in the presence of excess NO. This model revealed that the active and allosteric sites show cooperativity. This new comprehensive model gives a more accurate description of sGC regulation by NO and nucleotides in vivo.
authors.affiliation: Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037.
authors.forename: Nur Başak
authors.initials: NB
authors.lastname: Sürmeli
authors.affiliation: Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, 3584 CG Utrecht, The Netherlands.
authors.forename: Frederike M
authors.initials: FM
authors.lastname: Müskens
authors.affiliation: Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037. Electronic address: marletta@scripps.edu.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Marletta
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D004789:Enzyme Activation; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D020125:Mutation, Missense; D009569:Nitric Oxide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells; D000071756:Soluble Guanylyl Cyclase; D018411:Spodoptera
chemical_list: D018160:Receptors, Cytoplasmic and Nuclear; D009569:Nitric Oxide; D006418:Heme; D000255:Adenosine Triphosphate; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase
keywords: nitric oxide, guanylate cyclase (guanylyl cyclase), cyclic GMP (cGMP), ATP, signal transduction, heme