Kinetics of Calcium Release from Manganese Peroxidase during Thermal Inactivation

It was previously reported that manganese peroxidase from the white-rot fungusPhanerochaete chrysosporiumwas 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 10−2and 10−4M 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.