Effects of pH and sulfhydryl specific reagents on 4-fumarylacetoacetate fumarylhydrolase.
The pH-dependence of fumarylacetoacetase (4-fumarylacetoacetate fumaryl-hydrolase, EC 3.7.1.2) activity was studied in the pH range 6.25-8.50. After correction of the substrate concentration for enolate formation, the Michealis constant was found to be pH independent in this range. Likewise, the Ki values for the competitive inhibitors chloride and fluoride were found to be independent of pH between 6.25-8.50. A bell-shaped curve described the log V vs. pH dependence, and ionization constants of 6.5 and 8.2 were calculated. Tentatively an imidazole group and a sulfhydryl group were assigned to the constants 6.5 and 8.2, respectively. Both p-hydroxymercuribenzoate and 5,5'-dithiobis(2-nitrobenzoic acid) react with both sulfhydryl groups per subunit in the native protein and three sulfhydryl groups per subunit in the denatured protein. Substrate protects one sulfhydryl group in the native protein from reaction with 5,5'-dithiobis(2-nitrobenzoid acid). Substrate or the competitive inhibitor, fluoride, protect the enzyme from inactivation by p-hydroxymercuribenzoate. In addition p-hydroxymercuribenzoate shows saturation kinetics. Neither sulfhydryl inhibitor completely inactivates the enzyme. The enzyme is described as having three sulfhydryl groups per subunit, one of which is inaccessible to the sulfhydryl specific reagents when the protein is in the native state. One of the two accessible sulfhydryl groups is either near the active site or essential in maintaining the structure of the protein.
D000090:Acetoacetates / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D002417:Cattle; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005650:Fumarates / Q000037:antagonists & inhibitors / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D000090:Acetoacetates; D005650:Fumarates; D006902:Hydroxymercuribenzoates; D013439:Sulfhydryl Reagents; D004228:Dithionitrobenzoic Acid; D006867:Hydrolases; C020156:fumarylacetoacetase