Synthesis and evaluation of substrate analogues as mechanism-based inhibitors of type II isopentenyl diphosphate isomerase

Type 2 isopentenyl diphosphate isomerase (IDI-2), which catalyzes the interconversion of isopentenyl diphosphate and dimethylallyl diphosphate, contains a tightly bound molecule of FMN. To probe the mechanism of the reaction, cyclopropyl and epoxy substrate analogues, designed to be mechanism-based irreversible inhibitors, were synthesized and evaluated with IDI-2 from Thermus thermophilus. The cyclopropyl analogues were alternative substrates. The epoxy analogue was an irreversible inhibitor, with kI = 0.37 +/- 0.07 min(-1) and KI = 1.4 +/- 0.3 microM. LC-MS studies revealed formation of an epoxide-FMN adduct.