Abstract
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 kcat from 1400 to 3930 s-1 and the decrease in Km 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-order kinetics with an activation enthalpy (DeltaH*) of 191 kJ/mol while the heat denaturation process had a DeltaH* 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 Tm of 56 degrees C, which is much lower than that of the heat denaturation (Tm = 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.
Highlights
It is generally considered that a folded protein takes a unique conformation with a global or local minimum free energy state that is kinetically accessible [1,2,3,4,5]
The newly obtained low-activity form was brought to a highactivity 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
No direct evidence has been presented, the difference in the thermal stability as well as that in some kinetic properties between the catalytic reaction of the two active forms of carbamoylphosphate synthetase produced at different temperatures suggests that the enzyme adopts at least two active conformations [12]
Summary
Materials—Escherichia coli UM228, a catalase HPI-deficient mutant [17], was kindly provided by Dr P. Reaction was recorded as the decrease in absorbance at 240 nm, and the reaction rate was calculated from the maximum slope of the trace using the molar absorption coefficient of H2O2 of 43.6 MϪ1 cmϪ1 [20]. The subunit concentration of holocatalase I was measured spectrophotometrically at 406 nm using a molar absorption coefficient of 1.14 ϫ 105 MϪ1 cmϪ1 [21]. Heme content of purified catalase I was calculated from the values of absorbances at 280 and 406 nm of a sample solution using the following molar absorption coefficients [21]: 1.97 ϫ 105 MϪ1 cmϪ1 at 280 nm for apocatalase I; 2.18 ϫ 105 MϪ1 cmϪ1 at 280 nm and 1.14 ϫ 105 MϪ1 cmϪ1 at 406 nm for holocatalase I.
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