BIOPHYSICAL THEORY AND MODELING |
Structural Stabilization and Functional Improvement of
Horseradish Peroxidase upon Modification of Accessible
Lysines: Experiments and Simulation
Navid Mogharrab 1, Hedayatollah Ghourchian 1* and Mehriar Amininasab 2
1 Institute of Biochemistry & Biophysics, University of Tehran
2 Department of Cell and Molecular Biology, Faculty of Science, University of Tehran
* To whom correspondence should be addressed. E-mail: hadi{at}ibb.ut.ac.ir.
Submitted on July 12, 2006
Revised on August 14, 2006
Accepted on 20 October 2006
 |
Abstract |
|---|
Horseradish peroxidase (HRP) is an important heme enzyme with enormous medical diagnostic, biosensing and biotechnological applications. Thus, any improvement in the applicability and stability of the enzyme is potentially interesting. We previously reported that covalent attachment of an electron relay (anthraquinone 2-carboxylic acid) to the surface-exposed Lys residues successfully improves electron transfer properties of HRP. Here we investigated structural and functional consequences of this modification which alters three accessible charged lysines (Lys174, Lys232 and Lys241) to the hydrophobic anthraquinolysine residues. Thermal denaturation and thermoinactivation studies demonstrated that this kind of modification enhances the conformational and operational stability of HRP. The melting temperature increased 3 °C and the catalytic efficiency enhanced by 80%. Fluorescence and circular dichroism investigations suggest that the modified HRP benefits from enhanced aromatic packing and more buried hydrophobic patches as compared to the native one. Molecular dynamics simulations showed that modification improves the accessibility of His42 and heme prosthetic group to the peroxide and aromatic substrates, respectively. Additionally, the hydrophobic patch which functions as a binding site or trap for reducing aromatic substrates is more extended in the modified enzyme. In summary, this modification produces a new derivative of HRP with enhanced electron transfer properties, catalytic efficiency and stability for biotechnological applications.
Key Words:
anthraquinolysine, anthraquinone 2-carboxylic acid, chemical modification, horseradish peroxidase, molecular dynamic simulations
