Biochemical and pharmacological characterization of P-site inhibitors on homodimeric guanylyl cyclase domain from natriuretic peptide receptor-A.
Guanylyl cyclases catalyze the formation of cGMP from GTP. This family of enzymes includes soluble (sGC) and particulate guanylyl cyclases (pGC). The sGC are heterodimers containing one active catalytic site and one inactive pseudo-site. They are activated by nitric oxide. The pGC are homodimers whose activity is notably regulated by peptide binding to the extracellular domain and by ATP binding to the intracellular kinase homology domain (KHD). The catalytic mechanism of the pGC is still not well understood. Homology modeling of the structure of the homodimeric guanylyl cyclase domain, based on the crystal structure of adenylyl cyclase, suggests the existence of two functional sites for the substrate GTP. We used a purified and fully active recombinant catalytic domain from mammalian pGC, to document its enzyme kinetics properties in the absence of the KHD. The enzyme presents positive cooperativity with the substrate Mg-GTP. However, a heterodimeric catalytic domain mutant (GC-HET) containing only one active catalytic site is non-cooperative and is more similar to sGC. Structure-activity studies of purine nucleoside analogs indicate that 2'd3'GMP is the most potent inhibitor of pGC tested. It displays mixed non-competitive inhibition properties that are potentiated by the second catalytic product inorganic pyrophosphate (PPi). It appears to be equivalent to purinergic site (P-site) inhibitors characterized on particulate adenylyl cyclase. Inhibition of pGC by 2'd3'GMP in the presence of PPi is accompanied by a loss of cooperative enzyme kinetics. These results are best explained by an allosteric dimer model with positive cooperativity for both the substrate and inhibitors.
Joubert
Simon
S
Department of Pharmacology, Faculty of Medicine, Université de Montréal, Montréal, Québec, Canada H3T 1J4.
McNicoll
Normand
N
De Léan
André
A
D020816:Amino Acid Motifs / Q000187:drug effects*; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism; D001665:Binding Sites; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D007313:Insecta / Q000166:cytology; D011487:Protein Conformation; D018000:Receptors, Peptide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D018000:Receptors, Peptide; D009320:Atrial Natriuretic Factor; D006162:Guanylate Cyclase