Figure 1.

Biochemical studies of the Staphylococcus aureus IDI-2 enzyme. A) UV-visible absorption spectra of reduced IDI-2:FMN in the absence (blue) and presence (magenta) of IPP. The λ_max value of ~ 425 nm in the IDI-2:FMN:IPP complex is consistent with the neutral 1,5-dihydro-FMNH₂ tautomeric state of the reduced flavin. B) Pre-steady state accumulation and decay of the FMNH₂ intermediate under single turnover conditions. After the rapid accumulation phase (k_fast = 37 s⁻¹), the intermediate decays to an equilibrium level at a kinetically competent rate (k_slow = 2.1 s⁻¹ > k_cat = 1.3 s⁻¹), suggesting that the FMNH₂ species could be catalytically relevant. C) Pre-steady state burst experiment. The lack of a detectable pre-steady state burst in DMAPP formation suggests that the rate-determining step in the kinetic mechanism occurs prior to or concomitant with DMAPP formation, yielding a velocity (k_ss = 1.5 s⁻¹) that is very similar to k_cat (1.3 s⁻¹). This observation is consistent with rate-limiting isomerization chemistry step(s). Please note that the theoretical maximum burst amplitude of 1.0 would not be expected for the isomerization reaction catalyzed by IDI-2 where IPP and DMAPP would be in equilibrium at the active site. The depicted curve is only meant to illustrate the diagnostic utility of a pre-steady state burst experiment. D) An ¹H-NMR assay was used to measure a primary substrate deuterium kinetic isotope effect on the reaction velocity at saturating IPP concentrations as an approximation of ^Dk_cat. The observed isotope effect (^Dk_cat = 2.2) is consistent with cleavage of the IPP pro-R C2-H/D bond in a partially rate-determining step.