Thermal conversion from low- to high-activity forms of catalase I from Bacillus stearothermophilus

Catalase I from Bacillus stearothermophilus has the interesting property of increasing its enzyme activity on heating. It was confirmed that after heating at 70 degrees C for 10 min or 65 degrees C for 20 min, almost all the enzyme molecules were converted irreversibly to the activated form. The increase in k(cat) from 1400 to 3930 s(-1) and the decrease in K-m for H2O2 from 4.4 to 2.7 mM by heat activation indicate changes in the kinetic property of the enzyme molecule. Therefore, it follows that catalase I has two active forms, a high-activity form and a low-activity form. The heat activation process followed the first-or der kinetics with an activation enthalpy (Delta H*) of 191 kJ/mol while the heat denaturation process had a Delta H* of 545 kJ/mol. The CD spectra of the two enzyme forms had small but marked differences. The conversion of the low-activity form to the high-activity form was an endothermic process with a T-m of 56 degrees C, which is much lower than that of the heat denaturation (T-m = 76 degrees C), and the enthalpy change for the transition was only 5% of that for the denaturation. It has to be noted that the high-activity form of the enzyme was converted back to a low-activity form through the process of denaturation, refolding, and reconstitution with heme. In addition, the newly obtained low-activity form was brought to a high-activity form by heating. These results suggest that the native state of catalase I has two active conformations that are roughly the same but not identical and are separated by a high energy barrier.