Probable reaction mechanisms of flavokinase and FAD synthetase from rat liver

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Abstract

A steady-state kinetic analysis with evaluation of product inhibition was accomplished with purified rat liver flavokinase and FAD synthetase. For flavokinase, Km values were calculated as approximately 11 μm for riboflavin and 3.7 μm for ATP. Ki values were calculated for FMN as 6 μm against riboflavin and for ZnADP as 120 μm against riboflavin and 23 μm against ZnATP. From the inhibition pattern, the flavokinase reaction followed an ordered bi bi mechanism in which riboflavin binds first followed by ATP; ADP is released first followed by FMN. For FAD synthetase, Km values were calculated as 9.1 μm for FMN and 71 μm for MgATP. Ki values were calculated for FAD as 0.75 μm against FMN and 1.3 μm against MgATP and for pyrophosphate as 66 μm against FMN. The product inhibition pattern suggests the FAD synthetase reaction also followed an ordered bi bi mechanism in which ATP binds to enzyme prior to FMN, and pyrophosphate is released from enzyme before FAD. Comparison of Ki values with physiological concentrations of FMN and FAD suggests that the biosynthesis of FAD is most likely regulated by this coenzyme as product at the stage of the FAD synthetase reaction.

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      Once in the cell, Rf is converted into the flavin cofactor FAD via the sequential action of riboflavin kinase (ATP:riboflavin 5′ phosphotransferase, EC 2.7.1.26), which transfers a phosphoryl group from ATP to Rf to form FMN, and FAD synthase, previously known as FAD synthetase (FADS; ATP:FMN adenylyltransferase, EC 2.7.7.2), the enzyme responsible for FMN adenylation to FAD. In mammals, riboflavin kinase and FADS are two distinct monofunctional enzymes that have been purified from rat tissues and biochemically characterized (13–15). A controversial matter in FADS biochemistry is its subcellular localization.

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    This work was partly supported by NIH Grant DK 38940.

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