A point-mutated guanylyl cyclase with features of the YC-1-stimulated enzyme: Implications for the YC-1 binding site?

Guanylyl cyclases (GCs) and adenylyl cyclases (ACs) play key roles in vario us signaling cascades and are structurally closely related. The crystal str ucture of a soluble AC revealed one binding site each for the substrate ATP and the activator forskolin. Recently, YC-1, a novel activator of the hete rodimeric soluble GC (sGC), has been identified which acts like forskolin o n AC. Here, we investigated the respective substrate and potential activato r domains of sGC using point-mutated subunits. Whereas substitution of the conserved Cys-541 of the beta(1) subunit with serine led to an almost compl ete loss of activity, mutation of the respective homologue (Cys-596) in the alpha(1) subunit yielded an enzyme with an increased catalytic rate and hi gher sensitivity toward NO. This phenotype exhibits characteristics similar to those of the YC-1-treated wild-type enzyme. Conceivably, this domain wh ich corresponds to the forskolin site of the ACs may comprise the binding s ite for YC-1.