D N Kosarikov | Search PubMed

PubMed2001

Role of conformational changes in the heme-dependent regulation of human soluble guanylate cyclase.

Soluble guanylate cyclase (sGC) is a receptor for endogenous and exogenous nitric oxide (NO) and is activated many fold upon its binding, making it a core enzyme in the nitric oxide signal transduction pathway. Much effort has been made to understand the link between binding of NO at the sGC heme and activation of the cyclase activity. We report here the first direct evidence for the role of conformational changes in transmitting the signal between the heme and cyclase domains. Using both circular dichroism (CD) and fluorescence spectroscopies, we have probed the effect that the sGC activators NO and 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl-indazole (YC-1) and the inhibitor 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ) have on the structure of the protein. Surprisingly, binding of either ODQ or YC-1 to NO-bound sGC cause virtually identical changes in the far-UV CD spectra of sGC, reflecting a perturbation in the secondary structure of the enzyme. This change is absent upon binding of NO, YC-1 or ODQ alone. Using this and previous data, we propose a working model for the mechanism of activation of sGC by NO and YC-1 and inhibition by ODQ.

Acrylamide↗

PubMed2001

Human soluble guanylate cyclase: functional expression, purification and structural characterization.

Soluble guanylate cyclase is an enzyme that catalyzes formation of cGMP from GTP and is a member of the nucleotide cyclase family of enzymes. sGC is a receptor for endogenous and exogenous nitric oxide and is activated several-fold upon its binding, constituting a core enzyme in the nitric oxide signal transduction pathway. cGMP generated by sGC is an important second messenger that regulates activity of several enzymes triggering such important physiologic reactions as vasodilation, smooth muscle relaxation and platelet aggregation. We report here the functional expression of the human isoform of soluble guanylate cyclase in HighFive insect cells using a baculovirus expression system. Highly active recombinant protein was obtained without heme reconstitution or supplementation of the cell growth medium and the level of protein expression was found to be heavily affected by the composition of the growth medium. We have successfully purified highly active sGC (sp act up to 940 nmol/min/mg) from adherent cultures using a three-column, 1-day procedure. The UV-Vis spectrum of the isolated protein shows a Soret band at 431 nm, consistent with a histidine-ligated, 5-coordinate heme as previously reported. Far UV CD spectroscopy, intrinsic tryptophan fluorescence, fluorescence of the hydrophobic dye bis-ANS, size-exclusion chromatography, and small angle X-ray scattering (SAXS) were used to characterize the structural properties of the purified sGC. We used two hierarchical neural network methods to predict the secondary structure of sGC and found it to be consistent with the observed CD spectrum of sGC.

Amino Acid Sequence↗