Figure 8

LpxA T36N is less stable and less active than wild type LpxA. Continuous Fluorescence Enzyme Assay with Wild Type F. tularensis LpxA and LpxA T36N. (A) Graphs of the complete LpxA enzyme assay with wild type F. tularensis LpxA-His (dark green), LpxA-His T36N (dotted blue), and control reactions without, either nucleotide (purple), acyl-ACP (light green), or acyltransferase (black). (B) Graphs of the complete LpxA reaction after LpxA samples were frozen at −80°C. Wild type F. tularensis LpxA-His (dark green), LpxA-His T36N (dotted blue), and control reactions without, either nucleotide (purple), acyl-ACP (light green), or acyltransferase (black). The complete LpxA mixture contained 20 mM Hepes (pH 8.0), 40 μM 3-hydroxypalmitoyl-ACP, 4 mM UDP-GlcNAc, 10 nM F. tularensis LpxA-His₆ (added 5 minutes after ThioGlo) in a final volume of 100ul. The reaction was incubated at 25°C, and its progress was monitored continuously at λex = 379 nm and λem = 513 nm for 10 minutes at 15 second intervals. Control reactions were run in a similar fashion with the omission of substrate or enzyme as indicated. Each graph is a representative of three experiments.