title: Identification of the catalytic mechanism and estimation of kinetic parameters for fumarase.
abstract: The enzyme fumarase catalyzes the reversible hydration of fumarate to malate. The reaction catalyzed by fumarase is critical for cellular energetics as a part of the tricarboxylic acid cycle, which produces reducing equivalents to drive oxidative ATP synthesis. A catalytic mechanism for the fumarase reaction that can account for the kinetic behavior of the enzyme observed in both isotope exchange studies and initial velocity studies has not yet been identified. In the present study, we develop an 11-state kinetic model of the enzyme based on the current consensus on its catalytic mechanism and design a series of experiments to estimate the model parameters and identify the major flux routes through the mechanism. The 11-state mechanism accounts for competitive binding of inhibitors and activation by different anions, including phosphate and fumarate. The model is identified from experimental time courses of the hydration of fumarate to malate obtained over a wide range of buffer and substrate concentrations. Further, the 11-state model is found to effectively reduce to a five-state model by lumping certain successive steps together to yield a mathematically less complex representation that is able to match the data. Analysis suggests the primary reaction route of the catalytic mechanism, with fumarate binding to the free unprotonated enzyme and a proton addition prior to malate release in the fumarate hydration reaction. In the reverse direction (malate dehydration), malate binds the protonated form of the enzyme, and a proton is generated before fumarate is released from the active site.
authors.affiliation: Biotechnology and Bioengineering Center and Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
authors.forename: Muriel
authors.initials: M
authors.lastname: Mescam
authors.forename: Kalyan C
authors.initials: KC
authors.lastname: Vinnakota
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Beard
mesh_terms: D000818:Animals; D001671:Biochemistry / Q000379:methods; D002384:Catalysis; D020134:Catalytic Domain; D002952:Citric Acid Cycle; D005649:Fumarate Hydratase / Q000737:chemistry*; D015971:Gene Expression Regulation, Enzymologic; D006321:Heart / Q000502:physiology; D007700:Kinetics; D008293:Malates / Q000737:chemistry; D008956:Models, Chemical; D009206:Myocardium / Q000378:metabolism; D013552:Swine; D013816:Thermodynamics; D013997:Time Factors
chemical_list: D008293:Malates; C030298:malic acid; D005649:Fumarate Hydratase