title: Unbinding of hyaluronan accelerates the enzymatic activity of bee hyaluronidase.
abstract: Hyaluronan (HA), a polymeric glycosaminoglycan ubiquitously present in higher animals, is hydrolyzed by hyaluronidases (HAases). Here, we used bee HAase as a model enzyme to study the HA-HAase interaction. Located in close proximity to the active center, a bulky surface loop, which appears to obstruct one end of the substrate binding groove, was found to be functionally involved in HA turnover. To better understand kinetic changes in substrate interaction, binding of high molecular weight HA to catalytically inactive HAase was monitored by means of quartz crystal microbalance technology. Replacement of the delimiting loop by a tetrapeptide interconnection increased the affinity for HA up to 100-fold, with a K(D) below 1 nm being the highest affinity among HA-binding proteins surveyed so far. The experimental data of HA-HAase interaction were further validated showing best fit to the theoretically proposed sequential two-site model. Besides the one, which had been shown previously in course of x-ray structure determination, a previously unrecognized binding site works in conjunction with an unbinding loop that facilitates liberation of hydrolyzed HA.
authors.affiliation: Extracellular Matrix Research, Institute for Biomedical Aging Research, Austrian Academy of Sciences, A-6020 Innsbruck, Austria.
authors.forename: Attila
authors.initials: A
authors.lastname: Iliás
authors.affiliation: Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1518 Budapest, Hungary.
authors.forename: Károly
authors.initials: K
authors.lastname: Liliom
authors.affiliation: Extracellular Matrix Research, Institute for Biomedical Aging Research, Austrian Academy of Sciences, A-6020 Innsbruck, Austria.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Greiderer-Kleinlercher
authors.affiliation: Extracellular Matrix Research, Institute for Biomedical Aging Research, Austrian Academy of Sciences, A-6020 Innsbruck, Austria.
authors.forename: Stephan
authors.initials: S
authors.lastname: Reitinger
authors.affiliation: Extracellular Matrix Research, Institute for Biomedical Aging Research, Austrian Academy of Sciences, A-6020 Innsbruck, Austria. Electronic address: guenter.lepperdinger@oeaw.ac.at.
authors.forename: Günter
authors.initials: G
authors.lastname: Lepperdinger
mesh_terms: D000818:Animals; D001516:Bees / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006821:Hyaluronoglucosaminidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary
chemical_list: D019476:Insect Proteins; D006820:Hyaluronic Acid; D006821:Hyaluronoglucosaminidase