FIGURE 5.

Enzyme inhibition characterized by ITC single injection-type assays. (A) Inverse injection assay with α-amylase in the syringe and the substrate 2-chloro-4-nitrophenyl-maltoside (GalG₂CNP) in the syringe together with a variety of inhibitors: ACA (acarbose), CA (chlorogenic acid), EC (epicatechin), ECox (oxidized epicatechin), EGCG (epigallocatechin gallate), Mlv-3-glc (malvidin-3-glucoside) (Hanhineva et al., 2010). (B) MM/BH curves calculated from the curves in (A). (C) Single injection assay with substrate (benzoyl-L-arginine ethyl ester) and inhibitor (benzamidine) in the syringe and trypsin in the sample cell (Di Trani et al., 2018b). (D) K_m^app values extracted from direct fits to each of the injections (different colors) in (C). (E) Data from (C), deconvoluted using the empirical response model (Equation 10), converted to ν₀ and [S] and presented as a double-reciprocal plot. (F) Single injection assay with substrate (ATP) in the syringe and aminoglycoside-3′-phosphotransferase IIIa (APH) and kanamycin A in the sample cell (Wang et al., 2019). Under these dilute conditions [ATP] << K_m, ITC peaks decay exponentially with rate constant k_eff = k_cat/K_m. k_eff decreases with each injection due to product inhibition by ADP. (G) Plot of [APH]/k_eff as a function of total accumulated ADP concentration.