Mechanistic investigations of types I and II isopentenyl diphosphate isomerase

Isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI) catalyzes magnesium-dependent interconversion of the fundamental five-carbon isoprenoid building blocks: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Isomerization of IPP into DMAPP is an obligatory step in organisms that utilize the mevalonate (MVA) pathway for isoprenoid biosynthesis. Organisms that synthesize isoprenoids through the methylerythritol phosphate (MEP) pathway, however, generate IPP and DMAPP from a common metabolic precursor. Although these MEP-utilizing organisms often express IDI to balance substrates pools for chain elongation, the enzyme is non-essential.; Two forms of IDI are known. Type I IDI (IDI-1) was discovered in the 1950s, catalyzes a proton-addition elimination reaction through a carbocation intermediate, and requires divalent cations for enzymatic activity. A second unrelated IDI isoform, termed IDI-2, was discovered in 2001 that requires divalent cations and flavin mononucleotide (FMN) cofactor for activity. The mechanism of IDI-2 is currently unknown. Available evidence supports three possible mechanisms: proton addition-elimination similar to IDI-1, hydrogen atom addition elimination through a radical intermediate, or a sequential proton addition/single electron transfer (SET) mechanism.; The focus of this dissertation is to understand the catalytic mechanism of IDI-2 by testing substrate analogues as inhibitors and substrates, the use of kinetic and solvent isotope experiments, and progress curve analysis to explore stereochemical fidelity and proton exchange. Known inhibitors of IDI-1 are tested towards IDI-2 along with several novel substrate analogues containing radical clock moieties. The novel inhibitors are also tested with the IDI-1 enzymes from Rhodobacter capsulatus and Escherichia coli. These substrates provided additional insight into the catalytic mechanism of IDI-1.; The mechanistic studies reported in this dissertation provide compelling evidence that IDI-2 from Thermus thermophilus catalyzes isomerization of IPP and DMAPP by addition of a solvent-derived proton to the double bond of IPP to form a tertiary carbocation, similar to IDI-1. The intermediate carbocation may be stabilized through cation-pi interactions by the isoalloxazine ring of the flavin cofactor, analogous to a conserved tryptophan in IDI-1. The formation of covalent inhibitor-flavin adducts indicates that the flavin cofactor resides in proximity to the substrate and may serve as a general acid/base residue in the putative carbocationic mechanism. A catalytic mechanism involving radical intermediates, however, cannot yet be ruled out.