Inhibition of Deactivation of NO-sensitive Guanylyl Cyclase Accounts for the Sensitizing Effect of YC-1

Abstract

Many of the physiological effects of the signaling molecule nitric oxide are mediated by the stimulation of the NO-sensitive guanylyl cyclase. Activation of the enzyme is achieved by binding of NO to the prosthetic heme group of the enzyme and the initiation of conformational changes. So far, the rate of NO dissociation of the purified enzyme has only been determined spectrophotometrically, whereas the respective deactivation, i.e. the decline in enzymatic activity, has only been determined in cytosolic fractions and intact cells. Here, we report on the deactivation of purified NO-sensitive guanylyl cyclase determined after addition of the NO scavenger oxyhemoglobin or dilution. The deactivation rate corresponded to a half-life of the NO/guanylyl cyclase complex of ϳ4 s, which is in good agreement with the spectrophotometrically measured NO dissociation rate of the enzyme. The deactivation rate of the enzyme determined in platelets yielded a much shorter half-life indicating either partial damage of the enzyme during the purification procedure or the existence of endogenous deactivation accelerating factors. YC-1, a component causing sensitization of guanylyl cyclase toward NO, inhibited deactivation of guanylyl cyclase, resulting in an extremely prolonged half-life of the NO/guanylyl cyclase complex of more than 10 min. The deactivation of an ATP-utilizing guanylyl cyclase mutant was almost unaffected by YC-1, indicating the existence of a special structure within the catalytic domain required for YC-1 binding or for the transduction of the YC-1 effect. In contrast to the wild type enzyme, YC-1 did not increase NO sensitivity of this mutant, clearly establishing inhibition of deactivation as the underlying mechanism of the NO sensitizer YC-1.The signaling molecule nitric oxide (NO) has been shown to play an important role in the cardiovascular and nervous systems (1-4). Most of the effects of NO are mediated by the stimulation of the NO-sensitive guanylyl cyclase (GC), 1 the enzyme that catalyzes the formation of cGMP. The subsequent increase in cGMP leads to the activation of cGMP effector proteins like cGMP-activated protein kinases, cGMP-gated Ca 2ϩ channels and cGMP-regulated phosphodiesterases. NO-sensitive GC is a heterodimeric enzyme consisting of an ␣ and ␤ subunit (5). Although four enzyme subunits (␣ 1 , ␣ 2 , ␤ 1 , ␤ 2 ) have been identified so far, only the ␣ 1 ␤ 1 and ␣ 2 ␤ 1 heterodimers have been shown to exist as catalytically active NO sensitive isoforms in vivo (6). Each of the subunits contains the homologous cyclase catalytic domain. With two different cyclase catalytic domains the GTP-converting catalytic region of GC appears to be related to the ATP-converting catalytic region of the adenylyl cyclase (AC). The crystal structure of the catalytic center of AC has been resolved (7,8). In AC, the homologous cyclase catalytic domains C1 and C2 corresponding to the ␣ and ␤ subunits of NO-sensitive GC, respectively, are arranged in a "head-to-tail"-like orientation with two pseudosymmetric region...

Tonic and acute nitric oxide signaling through soluble guanylate cyclase is mediated by nonheme nitric oxide, ATP, and GTP

“…5). Furthermore, when YC-1 and excess NO are mixed with sGC, rapid deactivation with a NO trap does not occur, as reported (28). Given that YC-1 does not affect the rate of dissociation of NO from the sGC heme (S.P.L.C.…”

Kinetics of a Cellular Nitric Oxide/cGMP/Phosphodiesterase-5 Pathway

“…A plausible explanation is that the affinity of the receptors for NO differs in the two conditions. In cells, deactivation of GC upon the removal of NO was found to occur with a half-time of 0.2 s (7), which is about 10-fold faster than reported for the purified protein (29,30). Assuming that the deactivation rates reflect the rates of dissociation of NO from its receptors and that the association rates are the same, the receptor in cells would have a 10-fold lower affinity for NO, thereby accounting for the potency shift.…”

“…Assuming that the deactivation rates reflect the rates of dissociation of NO from its receptors and that the association rates are the same, the receptor in cells would have a 10-fold lower affinity for NO, thereby accounting for the potency shift. As to a possible mechanism, there is a site on the receptor protein whose pharmacological occupation leads to a reduction in the rate of deactivation and a corresponding increase in the potency of NO (29,30), so it is conceivable that there is an endogenous agonist for this site that does the reverse.…”

Functional Characterization of Two Nucleotide-binding Sites in Soluble Guanylate Cyclase

“…The NO-sensitizing action of YC-1 is due to the facil-itated formation of an active five-coordinate NO-heme (31). Furthermore, the sensitizing action of YC-1 is explained by preventing the release of NO from the active NO-heme (32). Similarly, the CO sensitization by YC-1 and BAY 41-2272 is associated with the formation of five-coordinate CO-heme (31,33).…”