Characterization and substrate specificity of fumarylacetoacetate fumarylhydrolase.
The molecular weight of fumarylacetoacetate fumarylhydrolase (EC 3.7.1.2) is 86 000 +/- 10 000, as determined by gel filtration. The enzyme appears to be a dimer with a monomer molecular weight of 38 000 - 43 000, as determined by gel electrophoresis, gel filtration in guanidine-hydrochloride, and ultracentrifugation. The subunits appear to be identical, as only one band is seen in gel electrophoresis, only one protein peak is detected in gel filtration in guanidine-hydrochloride, and only one amino-terminal amino acid (proline) is detected. Three free sulfhydryl groups per denatured monomer are detected by reaction with 5,5'-dithiobis(2-nitrobenzoic acid), while for the active enzyme only two sulfhydryl groups react with this reagent, The extinction coefficients at 260 and 280 nm, the amino acid composition, and the isoelectric point (6.7) of the enzyme are also reported. The enzyme catalyzes the hydrolysis of six 2,4-diketo acids and three 3,5-diketo acids tested. The Km of the substrates is similar but V varies by a factor of 120. The pH optimum is 7.3. The enzyme did not catalyze the hydrolysis of a number of esters tested.
D000090:Acetoacetates; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D005650:Fumarates; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000302:isolation & purification / Q000378:metabolism; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008970:Molecular Weight; D013329:Structure-Activity Relationship
D000090:Acetoacetates; D000596:Amino Acids; D005650:Fumarates; D046911:Macromolecular Substances; D006867:Hydrolases; C020156:fumarylacetoacetase