Co(II)-substituted Haemophilus influenzae β-carbonic anhydrase: Spectral evidence for allosteric regulation by pH and bicarbonate ion
| Autor: | Katherine van den Heever, Katherine M. Hoffmann, Dejan Samardzic, Roger S. Rowlett |
|---|---|
| Rok vydání: | 2011 |
| Předmět: |
Models
Molecular Absorption spectroscopy Dimer Bicarbonate Static Electricity Inorganic chemistry Allosteric regulation Biophysics chemistry.chemical_element Zinc Crystallography X-Ray Biochemistry Article chemistry.chemical_compound Allosteric Regulation Catalytic Domain Carbonic anhydrase Bicarbonate Ion Molecular Biology Carbonic Anhydrases Isosbestic point biology Cobalt Hydrogen-Ion Concentration Haemophilus influenzae Recombinant Proteins Bicarbonates Kinetics Crystallography chemistry Spectrophotometry biology.protein |
| Zdroj: | Archives of Biochemistry and Biophysics. 511:80-87 |
| ISSN: | 0003-9861 |
| DOI: | 10.1016/j.abb.2011.04.013 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect