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Mechanism of the Novel Prenylated Flavin-Containing Enzyme Ferulic Acid Decarboxylase Probed by Isotope Effects and Linear Free-Energy Relationships.
- Source :
-
Biochemistry . 5/24/2016, Vol. 55 Issue 20, p2857-2863. 7p. - Publication Year :
- 2016
-
Abstract
- 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 αβ 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; r² = 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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 00062960
- Volume :
- 55
- Issue :
- 20
- Database :
- Academic Search Index
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 115751394
- Full Text :
- https://doi.org/10.1021/acs.biochem.6b00170