title: Reaction-complexation coupling between an enzyme and its polyelectrolytic substrate: determination of the dissociation constant of the hyaluronidase-hyaluronan complex from the hyaluronidase substrate-dependence.
abstract: Hyaluronan (HA) is catalytically hydrolyzed by hyaluronidase (HAase). Depending on pH, HA is able to form a non-productive electrostatic complex with HAase in addition to the classical enzyme-substrate complex. Experiments have shown the strong inhibition of the HA hydrolysis catalyzed by HAase when performed at high HA over HAase concentration ratio and low ionic strength. The substrate-dependence thus shows a non-classic inhibition of HAase at high substrate concentrations due to the sequestration of HAase by HA in the electrostatic complex. The modeling of the HA/HAase system is characteristic of a reaction-complexation coupling and it is very difficult to study reaction or binding, separately. Here, we have established the equation controlling the global system and shown that the substrate-dependence of such a system is a direct combination of a pure Michaelis-Menten equation associated with the reaction and a hyperbolic curve associated with the binding. At low substrate concentrations, the hyperbola, representing the relative part of HAase not sequestered by HA, can be assimilated to a straight line. We have established the relationship between the slope of that straight line and the dissociation constant of the electrostatic HA-HAase complex. Fitting the theoretical equation to the experimental data allowed us to determine, for the first time, the Kd value of the non-productive HA-HAase complex at low ionic strength.
authors.affiliation: Laboratoire Polymères, Biopolymères, Surfaces, UMR 6270 CNRS - Université de Rouen, 76821 Mont-Saint-Aignan Cedex, France. helene.lenormand@univ-rouen.fr
authors.forename: Hélène
authors.initials: H
authors.lastname: Lenormand
authors.forename: Fériel
authors.initials: F
authors.lastname: Amar-Bacoup
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Vincent
mesh_terms: D006820:Hyaluronic Acid / Q000737:chemistry / Q000378:metabolism*; D006821:Hyaluronoglucosaminidase / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D009994:Osmolar Concentration; D011485:Protein Binding; D013379:Substrate Specificity
chemical_list: D006820:Hyaluronic Acid; D006821:Hyaluronoglucosaminidase