Publication Details (including relevant citation information):
WINGER, JA; MARLETTA, MA
BIOCHEMISTRY Volume: 44 Issue: 10 Pages: 4083-4090 Published: MAR 15 2005
The catalytic domains (alpha(cat) and beta(cat)) of alpha 1 beta 1 soluble guanylate cyclase (sGC) were expressed in Escherichia coli and purified to homogeneity. alpha(cat), beta(cat), and the alpha(cat)beta(cat) heterodimeric complex were characterized by analytical gel filtration and circular dichroism spectroscopy, and activity was assessed in the absence and presence of two different N-terminal regulatory heme-binding domain constructs. alpha(cat) and beta(cat) were inactive separately, but together the domains exhibited guanylate cyclase activity. Analysis by gel filtration chromatography demonstrated that each of the approximately 25-kDa domains form homodimers. Heterodimers were formed when alpha(cat) and beta(cat) were combined. Results from circular dichroism spectroscopy indicated that no major structural changes occur upon heterodimer formation. Like the full-length enzyme, the alpha(cat)beta(cat) complex was more active in the presence of Mn2+ as compared to the physiological cofactor Mg2+, although the magnitude of the difference was much larger for the catalytic domains than for the full-length enzyme. The Km for Mn2+-GTP was measured to be 85 +/- 18 mu M, and in the presence of Mn2+-GTP, the K-D for the alpha(cat)beta(cat) complex was 450 +/- 70 nM. The N-terminal heme-bound regulatory domain of the beta 1 subunit of sGC inhibited the activity of the alpha(cat)beta(cat) complex in trans, suggesting a domain-scale mechanism of regulation by NO. A model in which binding of NO to sGC causes relief of an autoinhibitory interaction between the regulatory heme-binding domain and the catalytic domains of sGC is proposed.
Address (URL): http://pubs.acs.org/doi/full/10.1021/bi047601d