Catalytic and thermodynamic properties of the urocanate hydratase reaction.
Urocanate hydratase (4-imidazolone-5-propionate hydro-lyase, EC 4.2.1.49) isolated from Pseudomonas putida contains covalently bound alpha-ketobutyrate as its cofactor. In the process of examining the mechanism by which alpha-ketobutyrate serves in this capacity, various thermodynamic parameters and temperature effects on urocanate hydratase activity were determined. As the equilibrium constant at 15 degrees C for imidazooone propionate formation from urocanate is approximately 69, regardless of whether urocanic acid or chemically synthesized imidazolone propionate is used as the initial substrate, it is concluded that the reaction is freely reversible. DeltaG degrees ', deltaH degrees ' and deltaS degrees ' were --2.5 kcal/mole, +5.2 kcal/mole and +26 cal/deg mole, respectively. Measurement of first-order reaction rates at various temperatures, in order to calculate the Arrhenius activation energy, showed a sharp break in the Arrhenius plot at 29 degrees C. Further examination of this phenomenon by determining s20,w values of urocanate hydratase as a function of temperature revealed a dramatic change at 31 degrees C. Since the enzyme in both experiments reverts to its original state when the temperature is lowered back below the transition point, it is proposed that urocanate hydratase undergoes a reversible conformational change or partial dissociation which affects its catalytic properties in the range of 29--31 degrees C.
D001665:Binding Sites; D002087:Butyrates; D006836:Hydro-Lyases; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles; D007651:Keto Acids; D007700:Kinetics; D011422:Propionates; D011485:Protein Binding; D011549:Pseudomonas / Q000201:enzymology; D013696:Temperature; D013816:Thermodynamics; D014559:Urocanate Hydratase / Q000302:isolation & purification / Q000378:metabolism
D002087:Butyrates; D007093:Imidazoles; D007651:Keto Acids; D011422:Propionates; D006836:Hydro-Lyases; D014559:Urocanate Hydratase