Thermodynamic study of magnesium ion binding to alpha-amylase.
The interaction of alpha-amylase (from Bacillus amyloliquefaciens) with Mg2+ ion was studied using UV spectrophotometric and isothermal titration calorimetric (ITC) methods at 27 degrees C 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 microM and 50 microM) of the protein. Alpha-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 (deltaH= -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.
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran. saboury@ut.ac.ir
Ali Akbar
AA
Saboury
Setareh
S
Ghasemi
Mohammad Umar
MU
Dahot
D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D002151:Calorimetry; D004795:Enzyme Stability; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D000516:alpha-Amylases / Q000737:chemistry / Q000378:metabolism*
D000516:alpha-Amylases; D008274:Magnesium