Chicken fumarase. II. Kinetic studies.
The catalysis of the hydration of fumarate and deshydration of L - malate by chicken fumarase was measured spectrophotometrically over a range of substrate concentrations from 4 times 10(-3) M to 8 times 10(-5) M for fumarate and from 8 times 10(-2) M to 10(-3) M for L - malate. For the forward and reverse reactions, linear Lineweaver and Burk plots were obtained. The Michaelis constants and the maximum initial velocities for both substrates were determined and the Haldane relation was found to be obeyed. The effect of pH on activity was investigated over a pH range from 5.5 to 9.0 and the data indicate the presence, in the active site, of two ionizable groups, one in the acidic form and one in the basic form. The values of the ionization constants, determined for the enzyme - substrate complexes, agree closely with the ones obtained for the porcine enzyme. The mode of action of twenty-four structural analogs on the initial velocity of the dehydration of L-malate, by chicken fumarase was examined. From these studies, two regions positively charged appear necessary for the effective binding of the carboxylates of the substrates and competitive inhibitors to the active center. Moreover, the data suggest the presence of an additional group, in the catalytic site of chicken fumarase, that stabilizes the carbon-carbon double bond common to fumarate and its structural analogs. Finally, from the comparison of the kinetic properties of the chicken and pig fumarases, it may be concluded that the catalytic mechanism of the homologous enzymes are very similar, if not identical.
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002645:Chickens / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005649:Fumarate Hydratase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005650:Fumarates / Q000378:metabolism; D006836:Hydro-Lyases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008293:Malates / Q000378:metabolism; D008954:Models, Biological; D009206:Myocardium / Q000201:enzymology*; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013386:Succinates / Q000378:metabolism; D013552:Swine; D013644:Tartrates / Q000494:pharmacology; D013816:Thermodynamics
D005650:Fumarates; D008293:Malates; D013386:Succinates; D013644:Tartrates; D006836:Hydro-Lyases; D005649:Fumarate Hydratase