Thermodynamic study of magnesium ion binding to ⍺-amylase

Abstract

The interaction of -amylase (from Bacillus amyloliquefaciens) with Mg²⁺ ion was studied using UV spectrophotometric and isothermal titration calorimetric (ITC) methods at 27ºC in 30 mM Tris buffer solution at pH = 7.0. The binding isotherm for metal-protein interaction was easily obtained by carrying out ITC experiment at two different concentrations (2 μM and 50 μM) of the protein. ⍺-Amylase had eight identical and independent binding sites for Mg²⁺ ion, which showed non-cooperativity in the binding process. The binding of Mg²⁺ ion was exothermic (ΔH= 17.3 kJ mol⁻¹) with association binding constant of 2.08 mM⁻¹. The binding slightly destabilized the enzyme against thermal denaturation, as evident from absorption studies.