FIGURE 4.

Enzyme kinetics of hIRE1α-cyto, mini-XBP-1 RNA stem-loop, and the inhibitor compound 3-ethoxy-5,6-dibromosalicylaldehyde. Titration of the substrate Cy5-labeled mini-XBP-1 RNA stem-loop as shown in Fig. 1, with constant enzyme (hIRE1α-cyto) concentration and measuring the slope of the initial reaction rates (A, i) determined the Michaelis-Menten constant (K_m). Using a Lineweaver-Burk plot (A, ii), the x-axis intercept therefore specified the K_m of 0.8 μm for a single mini-XBP-1 RNA stem-loop substrate (Fig. 1C). Titration of both substrate and 3-ethoxy-5,6-dibromosalicylaldehyde demonstrated a non-competitive mode of inhibition relative to the substrate (B). Data in B, i, were plotted as 1/velocity against 1/substrate concentration as a Lineweaver-Burk plot (B, ii). The binding constants K_ii and K_is were 71 and 88 nm, respectively. Data are shown from a representative of three separate experiments. Reaction kinetics data were fit using Visual Enzymics software.