title: Identification of residues crucially involved in soluble guanylate cyclase activation.
abstract: The ubiquitous heterodimeric nitric oxide (NO) receptor soluble guanylate cyclase (sGC) plays a key role in various signal transduction pathways. Binding of NO takes place at the prosthetic heme moiety at the N-terminus of the beta(1)-subunit of sGC. The induced structural changes lead to an activation of the catalytic C-terminal domain of the enzyme and to an increased conversion of GTP into the second messenger cyclic GMP (cGMP). In the present work we selected and substituted different residues of the sGC heme-binding pocket based on a sGC homology model. The generated sGC variants were tested in a cGMP reporter cell for their effect on the enzyme activation by heme-dependent (NO, BAY 41-2272) stimulators and heme-independent (BAY 58-2667) activators. The use of these experimental tools allows the enzyme's heme content to be explored in a non-invasive manner. Asp(44), Asp(45) and Phe(74) of the beta(1)-subunit were identified as being crucially important for functional enzyme activation. beta(1)Asp(45) may serve as a switch between different conformational states of sGC and point to a possible mechanism of action of the heme dependent sGC stimulator BAY 41-2272. Furthermore, our data shows that the activation profile of beta(1)IIe(145) Tyr is unchanged compared to the native enzyme, suggesting that Tyr(145) does not confer the ability to distinguish between NO and O(2). In summary, the present work further elucidated intramolecular mechanisms underlying the NO- and BAY 41-2272-mediated sGC activation and raises questions regarding the postulated role of Tyr(145) for ligand discrimination.
authors.affiliation: Cardiovascular Research, Bayer HealthCare, Aprather Weg 18a, D-42096 Wuppertal, Germany.
authors.forename: Christiane
authors.initials: C
authors.lastname: Rothkegel
authors.forename: Peter M
authors.initials: PM
authors.lastname: Schmidt
authors.forename: Friederike
authors.initials: F
authors.lastname: Stoll
authors.forename: Henning
authors.initials: H
authors.lastname: Schröder
authors.forename: Harald H H W
authors.initials: HH
authors.lastname: Schmidt
authors.forename: Johannes-Peter
authors.initials: JP
authors.lastname: Stasch
mesh_terms: D000818:Animals; D006152:Cyclic GMP / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000378:metabolism; D006418:Heme; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D051381:Rats; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D046968:Thermoanaerobacter / Q000201:enzymology
chemical_list: D006418:Heme; D006162:Guanylate Cyclase; D006152:Cyclic GMP