Jonathan Winger - Expression and characterization of the catalytic domains of soluble guanylate cyclase: Interaction with the heme domain

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      Publication Details (including relevant citation   information):

      WINGER, JA; MARLETTA, MA

      BIOCHEMISTRY Volume: 44 Issue: 10 Pages: 4083-4090 Published: MAR   15 2005

      Abstract:

      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