Co(II)-substituted Haemophilus influenzae β-carbonic anhydrase: spectral evidence for allosteric regulation by pH and bicarbonate ion.
Publication/Presentation Date
7-1-2011
Abstract
Cobalt(II)-substituted Haemophilus influenzae β-carbonic anhydrase (HICA) has been produced by overexpression in minimal media supplemented with CoCl(2), enabling kinetic, structural, and spectroscopic characterization. Co(II)-substituted HICA (Co-HICA) has comparable catalytic activity to that of wild-type enzyme with k(cat)=82±19 ms(-1) (120% of wild-type). The X-ray crystal structure of Co-HICA was determined to 2.5Å resolution, and is similar to the zinc enzyme. The absorption spectrum of Co-HICA is consistent with four-coordinate geometry. pH-dependent changes in the absorption spectrum of Co-HICA, including an increase in molar absorptivity and a red shift of a 580 nm peak with decreasing pH, correlate with the pH dependence of k(cat)/K(m). The absence of isosbestic points in the pH-dependent absorption spectra suggest that more than two absorbing species are present. The addition of bicarbonate ion at pH 8.0 triggers spectral changes in the metal coordination sphere that mimic that of lowering pH, supporting its hypothesized role as an allosteric inhibitor of HICA. Homogeneously (99±1% Co) and heterogeneously (52±5% Co) substituted Co-HICA have distinctly different colors and absorption spectra, suggesting that the metal ions in the active sites in the allosteric dimer of Co-HICA engage in intersubunit communication.
Volume
511
Issue
1-2
First Page
80
Last Page
87
ISSN
1096-0384
Published In/Presented At
Hoffmann, K. M., Samardzic, D., Heever, K.v, & Rowlett, R. S. (2011). Co(II)-substituted Haemophilus influenzae β-carbonic anhydrase: spectral evidence for allosteric regulation by pH and bicarbonate ion. Archives of biochemistry and biophysics, 511(1-2), 80–87. https://doi.org/10.1016/j.abb.2011.04.013
Disciplines
Medicine and Health Sciences
PubMedID
21531201
Department(s)
Fellows and Residents
Document Type
Article