Functional characterization of nitric oxide and YC-1 activation of soluble guanylyl cyclase: structural implication for the YC-1 binding site?
Soluble guanylyl cyclase (sGC) is a heterodimeric enzyme formed by an alpha subunit and a beta subunit, the latter containing the heme where nitric oxide (NO) binds. When NO binds, the basal activity of sGC is increased several hundred fold. sGC activity is also increased by YC-1, a benzylindazole allosteric activator. In the presence of NO, YC-1 synergistically increases the catalytic activity of sGC by enhancing the affinity of NO for the heme. The site of interaction of YC-1 with sGC is unknown. We conducted a mutational analysis to identify the binding site and to determine what residues were involved in the propagation of NO and/or YC-1 activation. Because guanylyl cyclases (GCs) and adenylyl cyclases (ACs) are homologous, we used the three-dimensional structure of AC to guide the mutagenesis. Biochemical analysis of purified mutants revealed that YC-1 increases the catalytic activity not only by increasing the NO affinity but also by increasing the efficacy of NO. Effects of YC-1 on NO affinity and efficacy were dissociated by single-point mutations implying that YC-1 has, at least, two types of interaction with sGC. A structural model predicts that YC-1 may adopt two configurations in one site that is pseudosymmetric with the GTP binding site and equivalent to the forskolin site in AC.
Lamothe
Maria
M
Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark, New Jersey 07103, USA.
Chang
Fu-Jung
FJ
Balashova
Nataliya
N
Shirokov
Roman
R
Beuve
Annie
A
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D006152:Cyclic GMP / Q000096:biosynthesis; D003545:Cysteine / Q000235:genetics; D003600:Cytosol / Q000201:enzymology; D004252:DNA Mutational Analysis; D004789:Enzyme Activation / Q000235:genetics; D020536:Enzyme Activators / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000737:chemistry; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D007191:Indazoles / Q000737:chemistry*; D007700:Kinetics; D008715:Methionine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000737:chemistry*; D010641:Phenotype; D021122:Protein Subunits / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011720:Pyrazoles / Q000737:chemistry; D011725:Pyridines / Q000737:chemistry; D051381:Rats; D012995:Solubility; D014443:Tyrosine / Q000235:genetics
C423582:3-(4-Amino-5-cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo(3,4-b)pyridine; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D007191:Indazoles; D021122:Protein Subunits; D011720:Pyrazoles; D011725:Pyridines; C090937:3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole; D009569:Nitric Oxide; D014443:Tyrosine; D001216:Asparagine; D008715:Methionine; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D003545:Cysteine