| Description |
Type 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) catalyzes the reversible conversion of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP). The enzyme requires a divalent cation (Mg2+) and a reduced flavin mononucleotide (FMNred) cofactor to be catalytically active. To probe the role of FMNred, substrate analogues were incubated with enzyme-bound FMNred and analyzed under various conditions by UV-vis absorption and fluorescence spectroscopy. The spectral characteristics indicated a covalent bond was formed between the analogues and flavin at either the C4a- or N5-position (dependent on the substrate analogues). Similar methodology was applied to study a flavin adduct that formed when IDI-2 was incubated with IPP or DMAPP. Furthermore, mass spectrometry demonstrated that this adduct was the result of a loss of inorganic diphosphate and yielded a single isoprene unit addition to flavin. Bond formation observed with many analogues and the natural substrates supports a role where flavin is involved with catalysis directly as a proton donor/acceptor and/or as a stabilizer of an intermediate. Additionally, the kinetic mechanism was established for the apo form of Streptococcus pneumoniae IDI-2, a potential antibacterial drug target. Bisubstrate studies (where IPP and FMN were varied) and competitive inhibition experiments (with a competitive inhibitor of both substrates) were employed to determine an ordered sequential mechanism, where FMN binds first. The kinetic data appeared sigmoidal and could only be fit when Hill coefficients where included in the mathematical models, particularly for FMN. The Hill coefficients were thoroughly evaluated statistically and their inclusion was determined to be significant, thus a possible cooperative or allosteric effect occurs upon the binding of FMN. These experiments suggest that allosteric inhibitors could be investigated to inactivate the S. pneumoniae IDI-2 enzyme. In a different application of statistics, transient kinetic isotope effect studies were evaluated using the bootstrap statistical technique. While statistically significant isotope effects were determined, the values were 100 % larger than expected. Thus, the technique was successful and identified an underlying problem with the experimental design during data collection. |
