Mechanism of Dimethylallyltryptophan Synthase: Evidence for a Dimethylallyl Cation Intermediate in an Aromatic Prenyltransferase Reaction

Dimethylallyltryptophan synthase is an aromatic prenyltransferase that catalyzes an electrophilic aromatic substitution reaction between dimethylallyl diphosphate (DMAPP) and L-tryptophan. The synthase is found in a variety of fungi, where it catalyzes the first committed step in the biosynthesis of the ergot alkaloids. The enzymatic reaction could follow either a dissociative mechanism involving a discrete dimethylallyl cation intermediate or an associative mechanism in which the indole ring directly displaces diphosphate in a single step. In this work, positional isotope exchange (PIX) experiments are presented in support of a dissociative mechanism. When [1-O-18]-DMAPP is subjected to the synthase reaction and recovered starting material is analyzed, 15% of the O-18-label is found to have scrambled from a bridging to a nonbridging position on the alpha-phosphorus. Kinetic isotope effect studies show that steps involved in the formation of the arenium ion intermediate are rate-determining, and therefore the scrambling occurs during the lifetime of the dimethylallyl cation/diphosphate ion pair. Similarly, when the unreactive substrate analogue, 6-fluorotryptophan, was employed, complete scrambling of the O-18-label. in DMAPP was observed. To our knowledge, this is the first observation of PIX in any prenyltransferase reaction, and it provides strong evidence supporting the existence of a carbocation intermediate.