Structure of AHAS. Schematic representation of the structure of yeast AHAS in complex with CS. active site of AHAS
Structure of monomer B in the Yeast-CE complex (Pang et al. J Biol Chem 2003) a , structure of monomer B in the yeast AHAS·CE complex. The amino acid residues that are not observed in the structure of the free enzyme are colored green (the mobile loop) and brown (the C-terminal arm). ThDP and FAD are displayed as tan-colored ball-and-stick models . b , major differences in the structure of yeast AHAS in the presence and absence of CE. The mobile loop region, FAD, and ThDP are shown in green (with CE) and blue (without CE). The C-terminal arm and CE are shown in brown.
Stereo diagram of the active site of AHAS II, based on the structure of the yeast AHASCIE complex (Protein Data Bank entry 1NOH) . The residues are labeled with the numbering of AHAS II, and residues belonging to the second catalytic polypeptide are marked with an asterisk . Thiamin diphosphate is shown with dark bonds , with its aminopyrimidine to the left and diphosphate to the right. C-2, the activated carbon, is labeled. Conformation A of Trp586 in Protein Data Bank entry 1N0H was used for Trp464.
Proposed structure for 2-ketobutyrate in the active site of the AHAS II-HEThDP intermediate. The angle of view is slightly different from that in Fig. 6, and peripheral residues have been eliminated so that the substrate can be visualized. A thin dotted red line connects the nucleophilic carbon of HEThDP and the carbonyl carbon of 2-ketobutyrate. Hydrogen bonds of interest are marked with dashed lines . B , schematic diagram with distances.
Stereo picture of the proposed structure for benzaldehyde in the active site of the AHAS II-HEThDP intermediate.
Overlay of sulfonylurea structures. The five yeast AHAS polypeptides (taken from complexes) were superimposed to reveal the relative orientations of the sulfonylurea herbicides.
herbicide binding site of AHAS. Distances in angstroms from CS are indicated with broken lines for hydrophobic contacts (black) and hydrogen bonds (blue). Distance cutoffs of 3.2 and 3.9 Å were employed for hydrogen bond and van der Waals interactions, respectively
Activity Test: Reaction vol. 200mL Reaction Mixture: 100mM Pyruvate, 1mm ThDP, 10mM Mg++ and 50mM FAD incubate 60 min at 37℃ Reaction stopped by 8N H 2 S04 0.5% creatine 5% α -naphthol ε =20000M- 1 cm- 1 of unknown color complex of acetolactate. 0.000042 (0.115% of wt) 69.66 mg 0.293U E85D AHAS 1.815 mg/mL (27mL) 0.000025 (0.068% of wt) 43.66 mg 0.475U E85A AHAS 2.15 mg/mL (27mL) 0.000365 14.8 mg 1.86U WT AHAS 0.866 mg/mL 0.165U Total Unit 0.000028 (0.077% of wt) 65.12 mg E85Q AHAS 2.22 mg/mL (20mL) Sp. Activity (U/mg) Total protein Protein conc. (vol)
E85A LB A600=0.604; IPTG 0.1mM 12 hrs induction at 18oC E85Q LB A600=0.567; IPTG 0.1mM 18hrs induction at 18oC BI AI Insoluble resin 20Kd M A1 A2 A3 A4 20Kd M BI AI insoluble Q1 Q3 Q2 Q4 FT FT Wash
<ul><li>Result of this week: </li></ul><ul><li>To increase solubility Triton X-100 (0.5%) used while elution </li></ul><ul><li>In order to remove high conc. Of NaCl, and Imidazole salt, purified proteins are dialyzed against 50mM TRIS pH8.0, 5mM EDTA and 1mM β ME and concentrated by Millipore (10YT). </li></ul><ul><li>Using G-10 desalting column protein became salt free (20mM TRIS pH 8.0) </li></ul><ul><li>In order to remove Triton X-100 we used Q-sepharose anion exchange column (elution buffer 20mM TRIS and 1M NaCl pH 8.0) </li></ul><ul><li>Protein conc. determined by Bradford method and it might be interfered with Triton X-100. </li></ul><ul><li>Purified Protein checked by 12% SDS-PAGE </li></ul>