AbstractThe SARS-CoV-2 Main protease (Mpro) is of major interest as an anti-viral drug target. Structure-based virtual screening efforts, fueled by a growing list of apo and inhibitor-bound SARS-CoV/CoV-2 Mprocrystal structures, are underway in many labs. However, little is known about the dynamic enzyme mechanism, which is needed to inform both structure-based design and assay development. Here, we apply Biodynamics theory to characterize the structural dynamics of substrate-induced Mproactivation, and explore the implications thereof for efficacious inhibition under non-equilibrium conditions. The catalytic cycle (including tetrahedral intermediate formation and hydrolysis) is governed byconcerteddynamic structural rearrangements of domain 3 and the m-shaped loop (residues 132-147) on which Cys145 (comprising the thiolate nucleophile and one-half of the oxyanion hole) and Gly143 reside (comprising the other half of the oxyanion hole). In particular:Domain 3 undergoes dynamic rigid-body rotations about the domain 2-3 linker, alternately visiting two conformational states (denoted as).The Gly143-containing crest of the m-shaped loop (denoted as crest B) undergoes up and down translations in concert with the domain 3 rotations (denoted as, whereas the Cys145-containing crest (denoted as crest A) remains statically in the up position. The crest B translations are driven by conformational transitions within the rising leg of the loop (Lys137-Asn142).We propose that substrates associate to thestate, which promotes thestate, dimerization (denoted as-substrate), and catalysis. The structure resets to the dynamic monomeric form upon dissociation of the N-terminal product. We describe the energetics of the aforementioned state transitions, and address the implications of our proposed mechanism for efficacious Mproinhibition under native-like conditions.