Mechanism of the Novel Prenylated Flavin-Containing Enzyme Ferulic Acid Decarboxylase Probed by Isotope Effects and Linear Free-Energy Relationships

Ferulic acid decarboxylase from Saccharomyces cerevisiae catalyzes the decarboxylation of phenylacrylic acid to form styrene using a newly described prenylated flavin mononucleotide cofactor. A mechanism has been proposed, involving an unprecedented 1,3-dipolar cyclo-addition of the prenylated flavin with the α═β bond of the substrate that serves to activate the substrate toward decarboxylation. We measured a combination of secondary deuterium kinetic isotope effects (KIEs) at the α- and β-positions of phenylacrylic acid together with solvent deuterium KIEs. The solvent KIE is 3.3 on Vmax/KM but is close to unity on Vmax, indicating that proton transfer to the product occurs before the rate-determining step. The secondary KIEs are normal at both the α- and β-positions but vary in magnitude depending on whether the reaction is performed in H2O or D2O. In D2O, the enzyme catalyzed the exchange of deuterium into styrene; this reaction was dependent on the presence of bicarbonate. This observation implies tha...