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-(18)O]-DMAPP is subjected to the synthase reaction and recovered starting material is analyzed, 15% of the (18)O-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 (18)O-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.
Luk
Louis Y P
LY
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T-1Z1, Canada.
Tanner
Martin E
ME
D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D000498:Allyl Compounds / Q000378:metabolism; D004122:Dimethylallyltranstransferase / Q000378:metabolism; D045782:Hemiterpenes / Q000378:metabolism; D008956:Models, Chemical; D019906:Nuclear Magnetic Resonance, Biomolecular; D009943:Organophosphorus Compounds / Q000378:metabolism
D000498:Allyl Compounds; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; C043060:3,3-dimethylallyl pyrophosphate; D019883:Alkyl and Aryl Transferases; D004122:Dimethylallyltranstransferase; C032409:tryptophan dimethylallyltransferase