Date of this Version
The Journal of Biological Chemistry VOL. 284, NO. 14, pp. 9433–9442, April 3, 2009
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 Km
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