Stereochemical Studies of the Type II Isopentenyl Diphosphate–Dimethylallyl Diphosphate Isomerase Implicate the FMN Coenzyme in Substrate Protonation

Abstract

The type II isopentenyl diphosphate: Dimethylallyl diphosphate isomerase (IDI‐2) is a flavoenzyme catalyzing the interconversion of IPP and DMAPP (see scheme). Chiral methyl analysis and proton inventory studies were used to probe the stereochemical course of this reaction. The results suggest that FMN acts as both the acid and base in IDI‐2 catalysis.

The type II isopentenyl Diphosphate:Dimethylallyl diphosphate isomerase (IDI-2): A model for acid/base chemistry in flavoenzyme catalysis

“…The observed IPP binding mode is consistent with stereochemical studies in that the pro-R C2 proton of IPP (which is removed during turnover) [31] is oriented towards the flavin. Moreover, in the observed binding mode, protonation at C4 of the substrate by the flavin would result in a suprafacial 1,3-proton migration – an observation that is consistent with the stereochemical outcome observed in a chiral methyl analysis of the IDI-2 catalyzed reaction [51]. In this study, deuterium (from D 2 O) was incorporated into (E )- and ( Z )-[4- 3 H]-IPP analogues ( 20 and 21 , Scheme 9) by IDI-2 under single turnover conditions to yield DMAPP products with a chiral ( E )-methyl group [51].…”

“…Moreover, in the observed binding mode, protonation at C4 of the substrate by the flavin would result in a suprafacial 1,3-proton migration – an observation that is consistent with the stereochemical outcome observed in a chiral methyl analysis of the IDI-2 catalyzed reaction [51]. In this study, deuterium (from D 2 O) was incorporated into (E )- and ( Z )-[4- 3 H]-IPP analogues ( 20 and 21 , Scheme 9) by IDI-2 under single turnover conditions to yield DMAPP products with a chiral ( E )-methyl group [51]. Each IPP analogue yielded the distinct enantiomeric configuration at the ( E )-methyl group of DMAPP that is consistent with protonation/deprotonation mediated by the flavin.…”

“…To test if proton transfer to C4 of IPP was also contributing to the rate-limiting step(s), solvent kinetic isotope effects and proton inventory studies were conducted by measuring the steady state kinetic parameters of IDI-2 as a function of D 2 O concentration [51]. The solvent KIEs of D2O k cat / K m = 3.8 and D2O k cat = 1.4 measured in 100% D 2 O suggest that bonds to solvent exchangeable protons are breaking in the transition state.…”

“…The solvent KIEs of D2O k cat / K m = 3.8 and D2O k cat = 1.4 measured in 100% D 2 O suggest that bonds to solvent exchangeable protons are breaking in the transition state. Moreover, the best fit of the proton inventory data (which in ideal circumstances can determine the contribution of individual solvent exchangeable protons to a solvent KIE) [52] was achieved with a model where a single solvent exchangeable proton is moving in a transition state that is only partially rate-limiting [51]. Altogether, these KIE studies are consistent with a chemical mechanism involving acid/base catalysis, perhaps involving proton transfers to and from the flavin (Scheme 7).…”

Methylerythritol Phosphate Pathway of Isoprenoid Biosynthesis

“…When epoxide and diene analogues of IPP ( 57, 58 , Figure 8B) were examined, time-dependent IDI-2 inactivation by covalent flavin modification at its C 4a position was observed (111, 115–117). Moreover, chiral methyl analysis of the DMAPP products derived from the IDI-2 catalyzed reaction with ( E )- and ( Z )-[4- 3 H]-IPP in D 2 O provides strong support for a chemical mechanism involving a flavin-mediated protonation at the vinyl C 4 position of the bound IPP substrate (118). Solvent kinetic isotope effect and proton inventory studies suggest that this proton transfer may be partially rate-limiting during steady state IDI-2 turnover (118).…”