Hyaluronidase activity of human Hyal1 requires active site acidic and tyrosine residues.
Hyaluronidases are a family of endolytic glycoside hydrolases that cleave the beta1-4 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 microm 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 V(max) 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.
Zhang
Ling
L
Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.
Bharadwaj
Alamelu G
AG
Casper
Andrew
A
Barkley
Joel
J
Barycki
Joseph J
JJ
Simpson
Melanie A
MA
D000143:Acids / Q000737:chemistry*; D055162:Biocatalysis; D020134:Catalytic Domain; D002460:Cell Line; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D006821:Hyaluronoglucosaminidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D000143:Acids; D004220:Disulfides; D009844:Oligosaccharides; D014443:Tyrosine; D006821:Hyaluronoglucosaminidase