Multiscale Modeling of Bovine Chymosin Catalysis: From AlphaFold3 and Replicated Molecular Dynamics to QM/MM Characterization of the Pre-Reactive State

Bovine chymosin B (Chn) selectively cleaves the Phe105–Met106 bond of κ-casein (κ-CN), initiating milk coagulation. The molecular organization of the enzyme–substrate complex immediately preceding cleavage remains incompletely characterized. Here, AlphaFold3-based structural modeling, molecular docking, replicated molecular dynamics (MD), and quantum mechanics/molecular mechanics (QM/MM) calculations were combined to characterize the pre-reactive Chn/κ-CN complex. Ten independent 300 ns MD trajectories were generated for each modeled system. Chn predominantly sampled an open Tyr77 conformation near 300°, while substrate binding was stabilized by a network of contacts involving the His-Pro region of κ-CN. The Phe105–Met106 scissile region remained locally mobile relative to the Asp34/Asp216 catalytic dyad, indicating a dynamically organized rather than rigid pre-reactive geometry. QM/MM refinement revealed a hydrogen-bond network involving Asp34, Asp216, the scissile peptide region, and several water molecules. An exploratory one-dimensional relaxed scan along the O(water)···C(carbonyl) coordinate revealed a non-monotonic potential-energy response, with a low-energy region at approximately 2.6–2.9 Å and an energetic increase upon further compression to 2.0 Å. These results provide an atomistic multiscale model of the pre-reactive Chn/κ-CN complex and identify structural features that may contribute to substrate recognition and catalytic organization in bovine chymosin.