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

Ling Zhang, Alamelu G. Bharadwaj, Andrew Casper, Joel Barkley, Joseph J. Barycki, Melanie A. Simpson

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

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 Glu 131 and Tyr 247 to Gln and Phe, respectively, eliminated activity at all hyaluronan concentrations (to 125 μM or 2.5 mg/ml). Conservative mutagenesis of Asp 129 and Tyr 202 significantly impaired catalysis by increases of 5- and 10-fold in apparent K m and reductions in Vmax of 95 and 50%, respectively. Tyr 247 and Asp 129 are required for stabilization of the catalytic nucleophile, which arises as a resonance intermediate of N-acetylglucosamine on the substrate. Glu 131 is a likely proton donor for the hydroxyl leaving group. Tyr 202 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 Asn 350 glycosylation as the requisite modification. Deletion of the C-terminal epidermal growth factor-like domain, in which Asn 350 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.

Original languageEnglish (US)
Pages (from-to)9433-9442
Number of pages10
JournalJournal of Biological Chemistry
Volume284
Issue number14
DOIs
StatePublished - Apr 3 2009

Fingerprint

Mutagenesis
Hyaluronoglucosaminidase
Hyaluronic Acid
Human Activities
Tyrosine
Catalytic Domain
Acetylglucosamine
Glycosylation
Catalysis
Enzymes
Molecular Probes
Glucuronic Acid
Substrates
Glycoside Hydrolases
Site-Directed Mutagenesis
Epidermal Growth Factor
Disulfides
Hydroxyl Radical
Enzyme kinetics
Nucleophiles

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Zhang, L., Bharadwaj, A. G., Casper, A., Barkley, J., Barycki, J. J., & Simpson, M. A. (2009). Hyaluronidase activity of human Hyal1 requires active site acidic and tyrosine residues. Journal of Biological Chemistry, 284(14), 9433-9442. https://doi.org/10.1074/jbc.M900210200

Hyaluronidase activity of human Hyal1 requires active site acidic and tyrosine residues. / Zhang, Ling; Bharadwaj, Alamelu G.; Casper, Andrew; Barkley, Joel; Barycki, Joseph J.; Simpson, Melanie A.

In: Journal of Biological Chemistry, Vol. 284, No. 14, 03.04.2009, p. 9433-9442.

Research output: Contribution to journalArticle

Zhang, L, Bharadwaj, AG, Casper, A, Barkley, J, Barycki, JJ & Simpson, MA 2009, 'Hyaluronidase activity of human Hyal1 requires active site acidic and tyrosine residues', Journal of Biological Chemistry, vol. 284, no. 14, pp. 9433-9442. https://doi.org/10.1074/jbc.M900210200
Zhang, Ling ; Bharadwaj, Alamelu G. ; Casper, Andrew ; Barkley, Joel ; Barycki, Joseph J. ; Simpson, Melanie A. / Hyaluronidase activity of human Hyal1 requires active site acidic and tyrosine residues. In: Journal of Biological Chemistry. 2009 ; Vol. 284, No. 14. pp. 9433-9442.
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