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Binding of Divalent Metal Ions to Calcium‐Free Peroxidase: Thermodynamic and Kinetic Studies
Author(s) -
Nazari Kohdadad,
Kelay Vahid,
Mahmoudi Ali,
Hashemianzadeh Seyed Majid
Publication year - 2012
Publication title -
chemistry and biodiversity
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.427
H-Index - 70
eISSN - 1612-1880
pISSN - 1612-1872
DOI - 10.1002/cbdv.201200043
Subject(s) - chemistry , cooperativity , divalent , metal , enthalpy , metal ions in aqueous solution , cooperative binding , horseradish peroxidase , crystallography , inorganic chemistry , binding site , enzyme , thermodynamics , organic chemistry , biochemistry , physics
Thermodynamics of binding of divalent metal ions including Ca 2+ , Mg 2+ , Ba 2+ , and Cd 2+ to Ca‐free horseradish peroxidase (HRP) enzyme was investigated using UV/VIS spectrophotometry and molecular‐mechanic (MM) calculations. According to the obtained binding and thermodynamic parameters, trend of the relative binding affinities of these divalent metal cations was found to be: Ca 2+ >Cd 2+ >Mg 2+ >Ba 2+ . Binding analysis based on Scatchard and Hill models showed positive cooperativity effect between the two distal and proximal binding sites. Furthermore, kinetics of binding and reconstitution process was examined (using relaxation‐time method) for binding of Ca 2+ (as the typical metal ion) to Ca‐free HRP, which was found a second‐order type having a two‐step mechanism involving fast formation of Ca‐free HRP/1 Ca 2+ as the kinetic intermediate in step 1. Finally, by means of MM calculations, the comparative stability energies were evaluated for binding of M 2+ metal cations to Ca‐free HRP. Based on MM calculations, preferential binding of Ca 2+ ion was occurred on distal and proximal binding sites of Ca‐free HRP associated with higher stability energies ( E total ). Indeed, among the divalent metal ions, Ca 2+ with the highest binding affinity (maximum value of K bin and minimum value of Δ G $\rm{{_{bin}^{0}}}$ ), maximum value of exothermic binding enthalpy, and stability energies stabilizes the HRP structure along with an optimized catalytic activity.