KINETICS OF CALCIUM-RELEASE FROM MANGANESE PEROXIDASE DURING THERMAL INACTIVATION

It was previously reported that manganese peroxidase from the white-ro t fungus Phanerochaete chrysosporium was susceptible to thermal inacti vation because it contains relatively labile Ca2+ ions required for st ability and activity [Sutherland and Aust (1996) Arch. Biochem. Biophy s. 332, 128-134], In this work we determined that four Ca2+ ions are p resent in the enzyme as isolated but this was reduced to 2 mol/mol upo n treatment with Ca2+-chelating agents or extensive dialysis of dilute enzyme. One of two relatively tightly bound Ca2+ remaining in the enz yme was released during thermal inactivation at pH 7.2. Inactive enzym e contained one Ca2+ which could be removed in acidic conditions. Inac tivation 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 af fect enzyme activity and did not seem to protect the enzyme from therm al inactivation at submicromolar enzyme concentrations. Excess Ca2+ or Mn2+ decreased the rate of the thermal inactivation and Mn2+ stabiliz ed the enzyme more efficiently than Ca2+ at higher temperature, Enzyme stabilization by Mn2+ was proposed to be due to binding of Mn2+ to th e Mn2+ substrate binding site. In competition studies, Ca2+ was shown to bind to this site with apparent dissociation constants of 10(-2) an d 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 s tabilize the enzyme by binding to both the Mn2+ site and, as previousl y proposed, to the distal Ca2+ site. (C) 1997 Academic Press.