Next, we tested if EDC can convert the transporter molecule, Cbz-D, into its anhydride state. In a vial, we fueled 10 mM Cbz-D with 100 mM EDC in 200 mM MES pH 6 and monitored the formation of the Cbz-D-anhydride in response to EDC by highperformance liquid chromatography (HPLC, Fig S2, Table ). We used a previously developed benzylamine quench assay to increase the accuracy. Using a kinetic model, we determined the reaction kinetics of the fuel conversion, activation, and deactivation and found that the anhydride half-life is 0.69 min (Fig. , Table ). Finally, we found no evidence of assembly before and after fueling by turbidity measurements (Fig S5 ).