Figure 2
From: Heme-dependent Inactivation of 5-Aminolevulinate Synthase from Caulobacter crescentus
Biochemical characterization, far-UV CD spectra, and thermal stability of cALAS. (A) The yellow (left) and red (right) fractions after anion exchange chromatography of recombinant cALAS. (B) Thin-layer chromatography of the reaction products of the ALA formation assays with the purified wild-type and mutants of recombinant cALAS. ALA (lane 1) and glycine (lane 2) standards; reaction mixtures with the PLP form of the wild-type (lane 3), the heme form of the wild-type (lane 4), the PLP forms of H340A (lane 5), C398A (lane 6), and the H340A/C398A double mutant (lane 7); reaction mixture without enzyme (lane 8); and a mixture of the ALA and glycine standards (lane 9). (C) Far-UV CD spectra of the PLP form (line 1) and the heme form (line 2) of cALAS were measured in 50 mM sodium phosphate buffer (pH 7.4) at 20 °C. The spectra were measured at protein concentrations of 7–10 μM in a 0.2 cm light-path cell. (D) Thermal scan profiles of the PLP form (line 1) and the heme form (line 2). Thermal denaturation of each form of cALAS was irreversible.
