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 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(kcat/KM) for the reaction correlated well with the Hammett σ(-) parameter with ρ = -0.39 ± 0.03; r(2) = 0.93. The negative ρ 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.
Department of Chemistry, ‡Department of Chemical Engineering, and §Department of Biological Chemistry, University of Michigan , Ann Arbor, Michigan 48109, United States.
Kyle L
KL
Ferguson
Department of Chemistry, ‡Department of Chemical Engineering, and §Department of Biological Chemistry, University of Michigan , Ann Arbor, Michigan 48109, United States.
Nattapol
N
Arunrattanamook
Department of Chemistry, ‡Department of Chemical Engineering, and §Department of Biological Chemistry, University of Michigan , Ann Arbor, Michigan 48109, United States.
E Neil G
EN
Marsh
D000179:Acrylates / Q000737:chemistry* / Q000378:metabolism; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000378:metabolism; D017368:Protein Prenylation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism
D000179:Acrylates; D029701:Saccharomyces cerevisiae Proteins; D002262:Carboxy-Lyases; C087194:phenylacrylic acid decarboxylase; C036658:acrylic acid