title: Functions of conserved cysteines of soluble guanylyl cyclase.
abstract: Soluble guanylyl cyclase (sGC), a heme-containing heterodimeric enzyme, is stimulated by NO and catalyzes the formation of the intracellular signaling molecule cGMP. Cysteine residues of sGC have been considered to be important as they were thought to play a significant role in the regulation of the enzyme. The aim of this study was to investigate the possible function of conserved cysteine residues of sGC. Fifteen conserved cysteine residues on sGC were point-mutated to serine, using site-directed mutagenesis. All of the resulting recombinant enzymes were able to synthesize cGMP. Mutation of two cysteines located in the N-terminal, putative heme-binding region of the beta1 subunit yielded proteins that were insensitive to NO. Spectrophotometric analysis of the NO-insensitive mutants purified from Sf9 cells revealed a loss of the prosthetic heme group. Both mutants could be reconstituted with heme and, as a consequence, NO sensitivity of the mutants was restored. Our data show that mutation of two cysteines of the beta1 subunit (Cys-78 and Cys-214) reduces the affinity of sGC for heme. Mutation of the corresponding cysteines on the alpha1 subunit did not alter NO responsiveness, indicating that heme-binding is mainly a feature of the N-terminal domain of the beta1 subunit.
authors.affiliation: Institut für Pharmakologie, Freie Universität Berlin, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Friebe
authors.forename: B
authors.initials: B
authors.lastname: Wedel
authors.forename: C
authors.initials: C
authors.lastname: Harteneck
authors.forename: J
authors.initials: J
authors.lastname: Foerster
authors.forename: G
authors.initials: G
authors.lastname: Schultz
authors.forename: D
authors.initials: D
authors.lastname: Koesling
mesh_terms: D000818:Animals; D019556:COS Cells; D003545:Cysteine; D004591:Electrophoresis, Polyacrylamide Gel; D006162:Guanylate Cyclase / Q000737:chemistry*; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000378:metabolism; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet
chemical_list: D009569:Nitric Oxide; D008345:Manganese; D006162:Guanylate Cyclase; D008274:Magnesium; D003545:Cysteine