Mechanism of Hyaluronic Acid Hydrolysis Catalyzed by Snake Venom Hyaluronidase

Hyaluronidases are widely distributed in nature being ubiquitous in snake species (svHyal). They catalyze the hydrolysis of β-1,4-glycosidic bonds in hyaluronic acid, a critical constituent of the extracellular matrix. This facilitates the spread of venom toxins into the bloodstream, exacerbating tissue damage and systemic toxicity─a rationale for their common designation as "spreading factors". While svHyals are not directly toxic, they substantially contribute to the morbidity and mortality associated with snakebite envenomation, the world's most lethal neglected tropical disease. Despite their important role in tissue penetration, the atomic-level reaction mechanism of these enzymes remains poorly understood. To bridge this knowledge gap, we studied the chemical mechanism of the Hyal-1 enzyme isolated from the Puff Adder viper (<i>Bitis arietans</i>), likely the major contributor to snakebite mortality in sub-Saharan Africa. We evaluated two alternative mechanistic scenarios, based on different protonation states for the active site "assisting residue" (Asp110), and conducted umbrella sampling QM/MM MD simulations (PBE/DZVP-GTH-PBE: AMBER). Our findings indicate that the pathway starting from a neutral Asp110 yields an activation free energy barrier of 20.34 kcal·mol^-1─nearly half that of the alternative pathway that considers an ionised Asp110. The deglycosylation step of the most favorable pathway yielded a free energy barrier of 13.94 kcal·mol^-1. Our simulations also support an induced-fit mechanism for the svHyal/hyaluronic acid complex, with substrate distortion (chair → boat/skew-boat) favoring a conformation that closely mimics the transition state. This distortion, along with a prealignment of Glu112, lowers the activation free energy, enhancing the susceptibility of the glycosidic bond to nucleophilic attack. The results are likely transferable to all svHyal given their high degree of interspecific similarity (>90% sequence identity). This study highlights the importance of understanding mechanistics, including detailed stereoelectronic conformations and subsite-specific interactions, for the design of novel and effective inhibitors with broad clinical and biotechnological applications.