Kinetics of calcium release from manganese peroxidase during thermal inactivation.
It was previously reported that manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium was susceptible to thermal inactivation because it contains relatively labile Ca2+ ions required for stability and activity [Sutherland and Aust (1996) Arch. Biochem. Biophys. 332, 128-134]. In this work we determined that four Ca2+ ions are present in the enzyme as isolated but this was reduced to 2 mol/mol upon treatment with Ca2+-chelating agents or extensive dialysis of dilute enzyme. One of two relatively tightly bound Ca2+ remaining in the enzyme was released during thermal inactivation at pH 7.2. Inactive enzyme contained one Ca2+ which could be removed in acidic conditions. Inactivation kinetics were biphasic and the rates for the two inactivation steps and the release of Ca2+ during inactivation suggested that the first, faster phase of inactivation was coupled to the removal of Ca2+. The weakly associated Ca2+ normally present in the enzyme did not affect enzyme activity and did not seem to protect the enzyme from thermal inactivation at submicromolar enzyme concentrations. Excess Ca2+ or Mn2+ decreased the rate of the thermal inactivation and Mn2+ stabilized the enzyme more efficiently than Ca2+ at higher temperature. Enzyme stabilization by Mn2+ was proposed to be due to binding of Mn2+ to the Mn2+ substrate binding site. In competition studies, Ca2+ was shown to bind to this site with apparent dissociation constants of 10(-2) and 10(-4) M at pH 4.5 and 7.2, respectively. Moreover, Ca2+ was a poor inhibitor of manganese peroxidase activity at pH 4.5. It is therefore suggested that Ca2+ is absent from the substrate site in physiological conditions but can bind to this site at higher pH and therefore may stabilize the enzyme by binding to both the Mn2+ site and, as previously proposed, to the distal Ca2+ site.
Timofeevski
S L
SL
Biotechnology Center, Utah State University, Logan 84322-4705, USA.
Aust
S D
SD
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D005658:Fungi / Q000201:enzymology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010544:Peroxidases / Q000378:metabolism*
D010544:Peroxidases; C051129:manganese peroxidase; D002118:Calcium