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 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.
Kobayashi
C
C
Department of Biotechnology, Faculty of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565, Japan.
Suga
Y
Y
Yamamoto
K
K
Yomo
T
T
Ogasahara
K
K
Yutani
K
K
Urabe
I
I
D002152:Calorimetry, Differential Scanning; D002374:Catalase / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D003067:Coenzymes; D004789:Enzyme Activation; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006358:Hot Temperature; D008956:Models, Chemical; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism
D003067:Coenzymes; D011994:Recombinant Proteins; C108078:catalase I; D002374:Catalase