YC-1 activation of human soluble guanylyl cyclase has both heme-dependent and heme-independent components

Abstract

YC-1 [3-(5-hydroxymethyl-2furyl)-1-benzyl indazole] is an allosteric activator of soluble guanylyl cyclase (sGC). YC-1 increases the catalytic rate of the enzyme and sensitizes the enzyme toward its gaseous activators nitric oxide or carbon monoxide. In other studies the administration of YC-1 to experimental animals resulted in the inhibition of the platelet-rich thrombosis and a decrease of the mean arterial pressure, which correlated with increased cGMP levels. However, details of YC-1 interaction with sGC and enzyme activation are incomplete. Although evidence in the literature indicates that YC-1 activation of sGC is strictly heme-dependent, this report presents evidence for both hemedependent and heme-independent activation of sGC by YC-1. The oxidation of the sGC heme by 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one completely inhibited the response to NO, but only partially attenuated activation by YC-1. We also observed activation by YC-1 of a mutant sGC, which lacks heme. These findings indicate that YC-1 activation of sGC can occur independently of heme, but that activation is substantially increased when the heme moiety is present in the enzyme.ODQ ͉ cGMP ͉ nitric oxide

Cobinamides Are Novel Coactivators of Nitric Oxide Receptor That Target Soluble Guanylyl Cyclase Catalytic Domain

“…Full-length sGC was purified from Sf9 cells as described previously (Martin et al, 2001). The quality of sGC preparations was assessed by the [␣-32 P]GTP 3 [ 32 P]cGMP conversion assay (see below) with 10 M DEA-NO donor.…”

“…Enzymatic activity was assayed using the [␣-32 P]GTP 3 [ 32 P] cGMP conversion assay as described previously (Martin et al, 2001) in a 100-l assay. The reaction is initiated by the addition of 1 mM GTP/[␣-32 P]GTP (ϳ100,000 cpm) to 0.1 g of sGC in 25 mM TEA, pH 7.5, 1 mg/ml bovine serum albumin, 1 mM 3-isobutyl-1-methylxanthine, 1 mM dithiothreitol, 1 mM cGMP, 3 mM MgCl 2 , 0.05 mg/ml creatine phosphokinase, and 5 mM creatine phosphate and incubated at 37°C for 10 min.…”

NO‐ and haem‐independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle

“…An indazole derivative, YC-1 has been described which stimulates sGC directly and sensitizes the enzyme towards its native activators NO and CO (Ko et al, 1994;Wu et al, 1995;Friebe et al, 1996;MuÈ lsch et al, 1997;Hoenicka et al, 1999). The exact mechanism of YC-1 dependent stimulation is not fully understood and is currently a subject of intense investigation (Sharma et al, 1999, Koesling, 2000Zhao et al, 2000;Martin et al, 2001).…”

Acrylamide Analog as a Novel Nitric Oxide-Independent Soluble Guanylyl Cyclase Activator

“…A recent report indicates that YC-1 activation of sGC can occur independently of heme, but that activation is substantially increased when the heme moiety is present in the enzyme (19). While addition of ODQ partially inhibited the stimulation of sGC by YC-1 (19) or A-778935, ODQ completely inhibited that by SNP, suggesting that A-778935 and NO activate sGC by different molecular mechanisms. ODQ binds to sGC in an NO-competitive manner and inhibits NO-stimulated activity by oxidizing the ferrous heme in the first complex to a five-liganded ferric intermediate.…”

“…Alternatively, YC-1 may stabilize the NO-sGC complex, decreasing the rate of dissociation (18). Activation of sGC by YC-1 occurs independently of heme, but is substantially increased in the presence of the heme moiety in the enzyme (19). Amino acids 259 -364 of the α 1 -subunit are the functional domain for the transduction of the NO activation signal and also represent the target for YC-1 binding (20).…”