Kinetic Analysis of Transient Intermediates in the Mechanism of Prenyl-Flavin-Dependent Ferulic Acid Decarboxylase.

Ferulic acid decarboxylase catalyzes the decarboxylation of various substituted phenylacrylic acids to their corresponding styrene derivatives and CO2 using the recently discovered cofactor prenylated FMN (prFMN). The mechanism involves an unusual 1,3-dipolar cycloaddition reaction between prFMN and the substrate to generate a cycloadduct capable of undergoing decarboxylation. Using native mass spectrometry, we show the enzyme forms a stable prFMN-styrene cycloadduct that accumulates on the enzyme during turnover. Pre-steady state kinetic analysis of the reaction using ultraviolet-visible stopped-flow spectroscopy reveals a complex pattern of kinetic behavior, best described by a half-of-sites model involving negative cooperativity between the two subunits of the dimeric enzyme. For the reactive site, the cycloadduct of prFMN with phenylacylic acid is formed with a kapp of 131 s-1. This intermediate converts to the prFMN-styrene cycloadduct with a kapp of 75 s-1. Cycloelimination of the prFMN-styrene cycloadduct to generate styrene and free enzyme appears to determine kcat for the overall reaction, which is 11.3 s-1.