title: The determination of enzyme-substrate dissociation rates by dynamic isotope exchange enhancement experiments.
abstract: A new method for the determination of dissociation rates of enzyme-substrate complexes has been developed. The rate of exchange of a labeled product back into the substrate is measured during catalysis of the forward reaction when the forward reaction is kept far from equilibrium by the enzymatic removal of the nonexchanging product. The ratio of the exchange rate and the net rate for product formation is then determined at various concentrations of the exchanging product. A plot of this ratio is a diagnostic indication of the kinetic mechanism and the relative rates of product dissociation from the binary and ternary enzyme complexes. This technique has been applied to the reaction catalyzed by bovine liver argininosuccinate lyase. The ratio for the rate of exchange of fumarate into argininosuccinate and the net rate for product formation was found to increase with the concentration of fumarate but to reach a limit of 3.3. The ratio of rates was half-maximal at 36 mM fumarate. The data have been interpreted to indicate the argininosuccinate lyase has a random kinetic mechanism. The calculated lower limit for the rate of release of arginine from the enzyme-fumarate-arginine complex is 0.35 times as fast as the Vmax in the reverse direction. The rate of release of arginine from the enzyme-arginine binary complex is 210 times faster than Vmax in the reverse direction.
mesh_terms: D000818:Animals; D001123:Argininosuccinate Lyase / Q000378:metabolism*; D001125:Argininosuccinic Acid / Q000378:metabolism; D002417:Cattle; D005650:Fumarates / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008190:Lyases / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D011865:Radioisotope Dilution Technique
chemical_list: D005650:Fumarates; D001125:Argininosuccinic Acid; C032005:fumaric acid; D008190:Lyases; D001123:Argininosuccinate Lyase