Purification and some properties of riboflavin synthetase from Bacillus stearothermophilus ATCC 8005.
A riboflavin synthetase was purified 51-fold from a thermophilic organism, Bacillus stearothermophilus ATCC 8005, that grew at 40 to 72 degrees C. Some of the properties of the enzyme are: (i) its temperature optimum was 95 degrees C, and the activity was negligible below 40 degrees C; (ii) the Arrhenius plot of the initial reaction rates was concave upward, with a break at 65 degrees C, and the apparent activation energies below and above 65 degrees C were 4.2 X 10(4) and 6.7 X 10(4) J/mol, respectively; (iii) the enzyme was fairly stable up to 60 degrees C without 6,7-dimethyl-8-ribityllumazine; this substance protected the enzyme from inactivation above 60 to 97 degrees C; (iv) the pH range for stability was 6.0 to 10.0 at 26 degrees C and 6.3 to 7.6 at 55 degrees C; (v) the enzyme was highly resistant at 26 degrees C to denaturation in 8 M urea, but the tolerance was extremely low at 55 degrees C; (vi) its molecular weight was estimated at 45,000; (vii) the Km for 6,7-dimethyl-8-ribityllumazine was 23 micrometer at 55 degrees C and 29 micrometer at 75 degrees C; (viii) its pH optimum was 6.7 to 7.2; (ix) 6-methyl-7-hydroxy-8-ribityllumazine was a competitive inhibitor (Ki = 0.18 micrometer); (x) the activity was sensitive to heavy-metal ions and thiol reagents; (xi) the enzyme did not require cofactor or a carbon donor; and (xii) the molar ratio of 6,7-dimethyl-8-ribityllumazine consumption to riboflavin formation was 2 throughout the entire reaction. Properties i through vi distinguish this enzyme from riboflavin synthetases purified by other investigators from mesophilic organisms, Ashbya gossypii, Eremothecium ashbyii, Escherichia coli, yeast, and spinach.
Purification and some properties of riboflavin synthetase from Bacillus stearothermophilus ATCC 8005. A riboflavin synthetase was purified 51-fold from a thermophilic organism, Bacillus stearothermophilus ATCC 8005, that grew at 40 to 72 degrees C. Some of the properties of the enzyme are: (i) its temperature optimum was 95 degrees C, and the activity was negligible below 40 degrees C; (ii) the Arrhenius plot of the initial reaction rates was concave upward, with a break at 65 degrees C, and the apparent activation energies below and above 65 degrees C were 4.2 X 10(4) and 6.7 X 10(4) J/mol, respectively; (iii) the enzyme was fairly stable up to 60 degrees C without 6,7-dimethyl-8-ribityllumazine; this substance protected the enzyme from inactivation above 60 to 97 degrees C; (iv) the pH range for stability was 6.0 to 10.0 at 26 degrees C and 6.3 to 7.6 at 55 degrees C; (v) the enzyme was highly resistant at 26 degrees C to denaturation in 8 M urea, but the tolerance was extremely low at 55 degrees C; (vi) its molecular weight was estimated at 45,000; (vii) the Km for 6,7-dimethyl-8-ribityllumazine was 23 micrometer at 55 degrees C and 29 micrometer at 75 degrees C; (viii) its pH optimum was 6.7 to 7.2; (ix) 6-methyl-7-hydroxy-8-ribityllumazine was a competitive inhibitor (Ki = 0.18 micrometer); (x) the activity was sensitive to heavy-metal ions and thiol reagents; (xi) the enzyme did not require cofactor or a carbon donor; and (xii) the molar ratio of 6,7-dimethyl-8-ribityllumazine consumption to riboflavin formation was 2 throughout the entire reaction. Properties i through vi distinguish this enzyme from riboflavin synthetases purified by other investigators from mesophilic organisms, Ashbya gossypii, Eremothecium ashbyii, Escherichia coli, yeast, and spinach.