Characterization and substrate specificity of fumarylacetoacetate fumarylhydrolase

Abstract

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.

Maleylacetoacetate Isomerase (MAAI/GSTZ)-Deficient Mice Reveal a Glutathione-Dependent Nonenzymatic Bypass in Tyrosine Catabolism

“…This biochemical phenotype was consistent with MAAI deficiency, because the FAH enzyme not only can hydrolyze FAA but also can convert SAA (formed from FAA and MAA) to succinic acid and acetoacetate and thereby prevent SA formation ( Fig. 1) (20,24). Previously the MAAI gene and MAAI activity were tested in patients manifesting "pseudotyrosinemia."…”

Mechanistic Inferences from the Crystal Structure of Fumarylacetoacetate Hydrolase with a Bound Phosphorus-based Inhibitor

“…Lysates were centrifuged, and recombinant FAH was purified from the supernatant using metal chelate chromatography by eluting with a solution containing 50 mM sodium phosphate, pH 6.0, 300 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 100 mM EDTA. FAH activity was assayed spectrophotometrically using acetopyruvate (AP) (Sigma Chemical Co.) and fumarylacetoacetate (FAA) prepared enzymatically from homogentisic acid (Sigma) as described previously (17,18,30). Assays were initiated by adding recombinant His-tagged FAH to reaction mixtures that covaried AP (0.625, 0.833, 1.25, 2.5, and 5.0 mM) with HMPOBA (0.0, 250, 500, and 750 M) or covaried FAA (3,8, and 15 M) with HMPOBA (0, 125, 250, 500, or 750 M).…”

“…FAH is a homodimer of 46 kDa subunits that catalyzes the hydrolytic cleavage of carboncarbon bonds in a variety of diketo acid substrates (18). In contrast to common biochemical hydrolysis reactions involving amide and ester bonds, hydrolytic cleavage of relatively stable carbon-carbon bonds is a fairly unusual reaction.…”

Type I Tyrosinemia: Lack of Immunologically Detectable Fumarylacetoacetase Enzyme Protein in Tissues and Cell Extracts

“…Fumarylacetoacetase purified from beef liver has been shown to exist of two identical monomers of a molecular weight approximately 43,000 (16). From Figure 2A it can be concluded that the human liver enzyme contains monomers of about the same molecular weight.…”

“…The method used for the isolation of fumarylacetoacetase from beef liver was based on that described by Mahuran et al (16). Unless otherwise specified, all the steps in the purification of the enzyme was camed out at 4" C. Centrifugation was canied out at 15,000 x g.…”