Enzymatic studies of the recombinant ALAS2 enzymes. (A) UV-visible absorption spectra (300-550 nm) of ALAS2 (WT) and Y586F2. Both proteins were at 2.0μM in 20mM HEPES, pH 7.5, containing 10% glycerol, and the spectra were recorded at 25°C. In both cases, the spectrum in gray is for the holoenzyme, whereas the black spectrum is for the holoenzyme in the presence of 100mM glycine. (B) Time courses for the reaction of WT ALAS2 (90μM; black) and Y586F (70μM; gray) with a mixture of glycine (100mM) and succinyl CoA (120μM) as monitored by following the changes in absorbance at 510 nm. The inset displays the first 5 seconds of the time courses. The collected data (dashed lines) were fitted to a 2-exponential equation with rate constants k₁ and k₂ for the formation and decay phases of the quinonoid intermediate (ie, 510-nm absorption species), respectively.²⁷  The rate constants for the WT ALAS2-catalyzed reaction are k₁ = 22.1 ± 1.1 seconds⁻¹ and k₂ = 0.57 ± 0.02 seconds⁻¹, whereas for the Y586F-catalyzed reaction are k₁ = 20.0 ± 3.2 seconds⁻¹ and k₂ = 1.11 ± 0.10 seconds⁻¹.