Biochemistry, Department of


Date of this Version



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


© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.


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

enzyme modulators.