Binding of Divalent Metal Ions to Calcium-Free Peroxidase: Thermodynamic and Kinetic Studies

作者：Nazari Kohdadad^*; Kelay Vahid; Mahmoudi Ali; Hashemianzadeh Seyed Majid

来源：Chemistry and Biodiversity, 2012, 9(9): 1806-1822.

DOI：10.1002/cbdv.201200043

• Horseradish peroxidase
• Divalent metal ions
• Kinetics
• Molecular-mechanics calculations

Thermodynamics of binding of divalent metal ions including Ca2+, Mg2+, Ba2+, and Cd2+ 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: Ca2+>Cd2+>Mg2+>Ba2+. 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 Ca2+ (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 Ca2+ as the kinetic intermediate in step 1. Finally, by means of MM calculations, the comparative stability energies were evaluated for binding of M2+ metal cations to Ca-free HRP. Based on MM calculations, preferential binding of Ca2+ 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, Ca2+ with the highest binding affinity (maximum value of K-bin and minimum value of Delta G(bin)(0)), maximum value of exothermic binding enthalpy, and stability energies stabilizes the HRP structure along with an optimized catalytic activity.

• 出版日期2012-9