A functional domain of the alpha1 subunit of soluble guanylyl cyclase is necessary for activation of the enzyme by nitric oxide and YC-1 but is not involved in heme binding.
Soluble guanylyl cyclase is a heterodimeric enzyme consisting of an alpha(1) and a beta(1) subunit and is an important target for endogenous nitric oxide and the guanylyl cyclase modulator YC-1. The activation of the enzyme by both substances is dependent on the presence of a prosthetic heme group. It has been unclear whether this prosthetic heme group is sandwiched between the alpha(1) and beta(1) subunits or whether it exclusively binds to the beta(1) subunit. Here we analyze progressive amino-terminal deletion mutants of the human alpha(1) subunit after co-expression with the human beta(1) subunit in the baculovirus/Sf9 system. Spectral, biochemical, and pharmacological analysis shows that the first 259 amino acids of the alpha(1) subunit can be deleted without loss of sensitivity to nitric oxide (NO) or YC-1 or loss of heme binding of the respective enzyme complex with the beta(1) subunit. This is in contrast to previous data indicating that NO sensitivity and a functional heme binding site requires full-length amino termini of bovine alpha(1) and beta(1) subunits. Further deletion of the first 364 amino acids of the alpha(1) subunit leads to an enzyme complex with preserved heme binding but loss of sensitivity to NO or YC-1 despite induction of the typical spectral shift by NO binding to the prosthetic heme group. We conclude that 1) the amino-terminal part of the alpha(1) subunit is not involved in heme binding and 2) amino acids 259-364 of the alpha(1) subunit represent an important functional domain for the transduction of the NO activation signal and likely represent the target for NO-sensitizing substances like YC-1.
Koglin
Markus
M
Institut für Experimentelle und Klinische Pharmakologie, Universität Hamburg, Martinistrasse 52, D-20246 Hamburg, Germany.
Behrends
Sönke
S
D000818:Animals; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000187:drug effects; D020536:Enzyme Activators / Q000494:pharmacology*; D017353:Gene Deletion; D006162:Guanylate Cyclase; D006418:Heme / Q000378:metabolism*; D006801:Humans; D007191:Indazoles / Q000494:pharmacology*; D009569:Nitric Oxide / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D000071756:Soluble Guanylyl Cyclase; D018411:Spodoptera
D020536:Enzyme Activators; D007191:Indazoles; D018160:Receptors, Cytoplasmic and Nuclear; C090937:3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole; D009569:Nitric Oxide; D006418:Heme; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase