Elucidation of the Catalytic Apparatus and Mechanism of Human Chitotriosidase‑1.

Despite extensive research over the past three decades, the catalytic mechanism of human chitotriosidase-1 (hCHIT1) has remained incompletely understood. To address this gap, we reanalyzed all available structural information and, integrating experimental data with multiscale molecular simulations, successfully modeled the full-length structure of hCHIT1 for the first time, including the previously unresolved proline-rich linker essential for domain communication. This comprehensive model enabled us to propose a general mechanism of hCHIT1 catalysis and to elucidate the distinct functional roles of all four highly conserved structural motifs of the glycoside hydrolase 18 (GH18) family, a group comprising over 65,000 known members across all domains of life. We further investigated the influence of monovalent metal ions in achieving optimal catalytic conditions and determined the activation energies for both substrate-assisted hydrolysis and transglycosylation processes. Our simulations revealed coordinated Brownian conformational fluctuations within hCHIT1 subdomains, which collectively harness thermal energy to drive catalysis. Notably, we discovered a previously unreported piston-like mechanism in which a conserved tyrosine residue transduces mechanical energy to the substrate, significantly lowering the activation barrier for catalysis. Additionally, by constructing a complete substrate model, we resolved the long-standing mechanistic enigma of the highly conserved tryptophan 'lid' at the active site entrance, demonstrating its multifaceted role in substrate gating, transition state stabilization, and product release. Finally, we demonstrated that binding of the first-in-class inhibitor OATD-01 induces subtle yet far-reaching dynamical changes within the active site, leading to dissociation of the immunoglobulin-like heterodimer and disruption of interactions with biological partners implicated in disease pathogenesis. These findings not only redefine the mechanistic landscape of hCHIT1 but also provide a robust framework for the rational design of next-generation GH18 inhibitors, for example, targeting multidrug-resistant pathogens.