Hyaluronidase activity of human Hyal1 requires active site acidic and tyrosine residues

Hyaluronidases are a family of endolytic glycoside hydrolases that cleave the β1-4 linkage between N-acetylglucosamine and glucuronic acid in hyaluronan polymers via a substrate-assisted mechanism. In humans, turnover of hyaluronan by this enzyme family is critical for normal extracellular matrix remodeling. However, elevated expression of the Hyal1 isozyme accelerates tumor growth and metastatic progression. In this study, we used structural information, site-directed mutagenesis, and steady state enzyme kinetics to probe molecular determinants of human Hyal1 function. Mutagenesis of active site residues Glu¹³¹ and Tyr²⁴⁷ to Gln and Phe, respectively, eliminated activity at all hyaluronan concentrations (to 125 μM or 2.5 mg/ml). Conservative mutagenesis of Asp¹²⁹ and Tyr²⁰² significantly impaired catalysis by increases of 5- and 10-fold in apparent K_m and reductions in Vmax of 95 and 50%, respectively. Tyr²⁴⁷ and Asp¹²⁹ are required for stabilization of the catalytic nucleophile, which arises as a resonance intermediate of N-acetylglucosamine on the substrate. Glu¹³¹ is a likely proton donor for the hydroxyl leaving group. Tyr²⁰² is a substrate binding determinant. General disulfide reduction had no effect on activity in solution, but enzymatic deglycosylation reduced Hyal1 activity in a time-dependent fashion. Mutagenesis identified Asn³⁵⁰ glycosylation as the requisite modification. Deletion of the C-terminal epidermal growth factor-like domain, in which Asn³⁵⁰ is located, also eliminated activity, irrespective of glycosylation. Collectively, these studies define key components of Hyal1 active site catalysis, and structural factors critical for stability. Such detailed understanding will allow rational design of enzyme modulators.