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 mono nucleotide cofactor. A mechanism has been proposed, involving an unprecedented 1,3-dipolar cyclo-addition of the prenylated flavin with the alpha=beta 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 alpha- and beta-positions of phenylacrylic acid together with solvent deuterium KIEs. The solvent Kill is 3.3 on V-max/K-M but is close to unity on V-max, indicating that proton transfer to the product occurs before the rate-determining step. The secondary KIEs are normal at both the alpha- and beta-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 that CO2 release must occur after protonation of the product. Further information was obtained from a linear free-energy analysis of the reaction through the use of a range of para- and meta-substituted phenylacrylic acids. Log(k(cat)/K-M) for the reaction correlated well with the Hammett sigma(-) parameter with rho = -0.39 +/- 0.03; r(2) = 0.93. The negative rho value and secondary isotope effects are consistent with the rate-determining step being the formation of styrene from the prenylated flavin product adduct through a cyclo-elimination reaction.