Mechanistic Studies of the Protonation-Deprotonation Reactions for Type 1 and Type 2 Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase

Abstract

Type 1 and type 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-1 and IDI-2) catalyze the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the fundamental building blocks for biosynthesis of isoprenoid compounds. Previous studies indicate that both isoforms of IDI catalyze isomerization by a protonation-deprotonation mechanism. IDI-1 and IDI-2 are "sluggish" enzymes with turnover times of ∼10 s^-1 and ∼1 s^-1, respectively. We measured incorporation of deuterium into IPP and DMAPP in D₂O buffer for IDI-1 and IDI-2 under conditions where newly synthesized DMAPP is immediately and irreversibly removed by coupling its release to condensation with l-tryptophan catalyzed by dimethylallyltrytophan synthase. During the course of the reactions, we detected formation of d₁, d₂, and d₃ isotopologues of IPP and DMAPP, which were formed during up to five isomerizations between IPP and DMAPP during each turnover. The patterns for deuterium incorporation into IPP show that d₂-IPP is formed in preference to d₁-IPP for both enzymes. Analysis of the patterns of deuterium incorporation are consistent with a mechanism involving addition and removal of protons by a concerted asynchronous process, where addition substantially precedes removal, or a stepwise process through a short-lived (<3 ps) tertiary carbocationic intermediate. Previous work with mechanism-based inhibitors and related model studies supports a concerted asynchronous mechanism for the enzyme-catalyzed isomerizations.