CATALYTIC PROPERTIES OF RIBOFLAVIN SYNTHE-TASE FROM A HIGH-RIBOFLAVINOGENIC EREMOTHECIUM ASHBYII
1974 Volume 20 Issue 4 Pages 301-316
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1974 Volume 20 Issue 4 Pages 301-316
Riboflavin synthetase catalyzes a conversion of two molecules of 6, 7-dimethyl-8-ribityllumazine to one molecule each of riboflavin and 4-ribitylamino-5-aminouracil. A riboflavinogenic Eremothecium ashbyii produces this enzyme only within an earlier stage of the growth phase of its fungal life cycle prior to an initiation of vitamin overproduction, and the enzyme persists to function throughout the cycle. The specific enzyme activity of the cell-free extract is high, 72 to 165 mμmoles of riboflavin formed per hour per mg of protein at 37°C, approximately 100 times higher than those of the vitamin-producing microbes examined to date.
The enzyme exhibited a broad plateau on its activity curve between pH of 5.7 and 7.0 with a maximum point at pH 6.5. The optimal activation with temperature occurred at 47°C, but the enzyme was completely inactivated at 60°C. The apparent activation energy was 11, 000 calories per mole between 42° and 20°C. The initial reaction velocity showed a second order dependency on the lumazine concentration in the range 1.5×10-4 to 6.3×10-6M, i, e., a sigmoidal kinetic response instead of a hyperbolic Michaelis-Menten behavior, suggesting that affinities of the two substrate binding sites present on the enzyme have similar orders of magnitude. A Hill plot of the velocity consists of two straight lines with a slope=2 at lumazine levels below 6.0×10-5M and with a slope=1 at the levels beyond this value. When the lumazines were bound to the enzyme in a random order, Ka'+Kd' and Ka'Kd (or KaKd'), where Ka' and Kd' denote the dissociation constants of a ternary enzyme-substrate complex and Ka and Kd are the constants of the two binary complexes present, were calculated as 4.5×10-5M and 2.7×10-9M2, respectively, while, when an ordered mechanism held, Kd and Ka' (or Ka and Kd') corresponded to the respective values of 6.0×10-5 and 4.5×10-5M. A reaction mechanism without a kinetically significant ternary complex was not the case for E. ashbyii enzyme.
A simplified method for purification of riboflavin synthetase from E. ashbyii is reported. The specific enzyme activity of the purified preparation was 6, 370mμmoles of the vitamin produced for one hour per mg of protein at 37°C.
