A constitutively activated mutant of human soluble guanylyl cyclase (sGC): implication for the mechanism of sGC activation.
Heterodimeric alphabeta soluble guanylyl cyclase (sGC) is a recognized receptor for nitric oxide (NO) and mediates many of its physiological functions. Although it has been clear that the heme moiety coordinated by His-105 of the beta subunit is crucial for mediating the activation of the enzyme by NO, it is not understood whether the heme moiety plays any role in the function of the enzyme in the absence of NO. Here we analyze the effects of biochemical and genetic removal of heme and its reconstitution on the activity of the enzyme. Detergent-induced loss of heme from the wild-type alphabeta enzyme resulted in several-fold activation of the enzyme. This activation was inhibited after hemin reconstitution. A heme-deficient mutant alphabetaCys-105 with Cys substituted for His-105 was constitutively active with specific activity approaching the activity of the wild-type enzyme activated by NO. However, reconstitution of mutant enzyme with heme and/or DTT treatment significantly inhibited the enzyme. Mutant enzyme reconstituted with ferrous heme was activated by NO and CO alone and showed additive effects between gaseous effectors and the allosteric activator 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine. We propose that the heme moiety through its coordination with His-105 of the beta subunit acts as an endogenous inhibitor of sGC. Disruption of the heme-coordinating bond induced by binding of NO releases the restrictions imposed by this bond and allows the formation of an optimally organized catalytic center in the heterodimer.
Martin
Emil
E
Department of Integrative Biology and Pharmacology and Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX 77030, USA.
Sharina
Iraida
I
Kots
Alexander
A
Murad
Ferid
F
D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005822:Genetic Vectors; D006162:Guanylate Cyclase; D006418:Heme / Q000737:chemistry; D006427:Hemin / Q000737:chemistry; D006801:Humans; D007313:Insecta; D007501:Iron / Q000378:metabolism; D008024:Ligands; D008956:Models, Chemical; D009154:Mutation; D009569:Nitric Oxide / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics* / Q000378:metabolism*; D000071756:Soluble Guanylyl Cyclase; D013053:Spectrophotometry; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D003902:Detergents; D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear; D009569:Nitric Oxide; D006418:Heme; D006427:Hemin; D007501:Iron; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D003545:Cysteine; D004229:Dithiothreitol