Substrate α-1,4-Glucan Structural Organization Determines EGCG Inhibition of α-Amylase through an Enzyme-Substrate-Inhibitor Tug-of-War.

α-Amylase inhibitors often exhibit markedly inconsistent activities across substrate systems, yet the molecular basis remains poorly understood. Here, using epigallocatechin gallate (EGCG) as a model inhibitor, we compared corn starch, a continuous α-1,4-glucan, with the small, structurally defined chromogenic substrate GalG2CNP, demonstrating that substrate structural organization, rather than the inhibitor itself, governs apparent inhibitory outcomes. Their distinct enzyme affinities produced opposing responses: low-affinity GalG2CNP was readily inhibited by EGCG, whereas high-affinity starch resisted inhibition even with excess inhibitor. Multimodal biophysics, molecular docking, and molecular dynamics simulations confirmed that EGCG occupies the α-amylase active pocket without substantially perturbing enzyme structure. Ternary system analyses further revealed that high-affinity starch displaced prebound EGCG, whereas GalG2CNP induced only partial re-equilibration. We therefore propose a substrate-affinity-driven tug-of-war model and a two-tier screening strategy, using artificial substrates for primary screening and starch for physiological verification, providing a mechanistic framework for more reliably evaluating candidate dietary α-amylase inhibitors.