Influence of calcium binding on the thermal stability of ‘thermitase’, a serine protease from Thermoactinomyces vulgaris

Abstract

‘Thermitase’ (EC 3.4.21.14), a thermostable extracellular serine protease from Thermoactinomyces vulgaris, binds one calcium ion with a dissociation constant of about 10⁻⁴ M at 25°C and pH 7.5 to 3.5. In addition, two calcium ions are bound more tightly to the enzyme, as shown by experiments with a calcium-selective electrode. The single most weakly bound calcium ion causes a slight quenching of the protein fluorescence emission, accompanied by a stabilization against thermal denaturation or autolysis and an increase of esterolytic activity of approx. 10%. The tightly bound calcium ions have only a slight influence on activity or on thermal denaturation or autolytic degradation. The activation parameters of thermal denaturation indicate that ‘thermitase’ belongs to the class of thermostable enzymes with a high intrinsic stability.