Figure 9.

(A) Time-dependence of [P] from Opt–2 cleavage by PLE (25 nM) at 37 °C. (B) Corresponding time-dependence of $\left[\text{A}\right]=\left[\text{I}\right]+\left[\text{P}\right]$. (C) Michaelis–Menten plot for the initial rates of ${\left[\text{A}\right]}_t$ formation as a function of [Opt–2]₀. A linear fit to eq 5 enabled the determination of the values of $k_{\text{cat}}\text{/}{K}_{\text{M}}$ (±95% CI) and a lower limit for $K_{\text{M}}$. Assays were performed in 10 mM HEPES–NaOH buffer, pH 7.4, containing DMF (1.5% v/v) and Triton X-100 (0.8% w/v). Gray areas represent the SD; n = 2 independent replicates, n = 3 technical replicates.