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Purification and Properties of a Folate-catabolizing Enzyme

2000; Elsevier BV; Volume: 275; Issue: 45 Linguagem: Inglês

10.1074/jbc.m005864200

1083-351X

Jae Rin Suh, Emia W. Oppenheim, Sameh Girgis, Patrick J. Stover,

Porphyrin Metabolism and Disorders

We have identified and purified to homogeneity an enzyme from rat liver that catalyzes the oxidative catabolism of 5-formyltetrahydrofolate to p-aminobenzoylglutamate and a pterin derivative. Purification of the enzyme utilized six column matrices, including a pterin-6-carboxylic acid affinity column. Treatment of crude rat liver extracts with EDTA or heat decreased the specific activity of the enzyme by up to 85%. Peptides generated from the purified protein were sequenced and found to be identical to primary sequences present within rat light chain or heavy chain ferritin. Commercial rat ferritin did not display catabolic activity, but activity could be acquired with iron loading. The purified enzyme contained 2000 atoms of iron/ferritin 24-mer and displayed similar electrophoretic properties as commercial rat liver ferritin. The ferritin-catalyzed reaction displayed burst kinetics, and the enzyme catalyzed only a single turnover in vitro. Expression of rat heavy chain ferritin cDNA resulted in increased rates of folate turnover in cultured Chinese hamster ovary cells and human mammary carcinoma cells and reduced intracellular folate concentrations in Chinese hamster ovary cells. These results indicate that ferritin catalyzes folate turnover in vitro and in vivoand may be an important factor in regulating intracellular folate concentrations. We have identified and purified to homogeneity an enzyme from rat liver that catalyzes the oxidative catabolism of 5-formyltetrahydrofolate to p-aminobenzoylglutamate and a pterin derivative. Purification of the enzyme utilized six column matrices, including a pterin-6-carboxylic acid affinity column. Treatment of crude rat liver extracts with EDTA or heat decreased the specific activity of the enzyme by up to 85%. Peptides generated from the purified protein were sequenced and found to be identical to primary sequences present within rat light chain or heavy chain ferritin. Commercial rat ferritin did not display catabolic activity, but activity could be acquired with iron loading. The purified enzyme contained 2000 atoms of iron/ferritin 24-mer and displayed similar electrophoretic properties as commercial rat liver ferritin. The ferritin-catalyzed reaction displayed burst kinetics, and the enzyme catalyzed only a single turnover in vitro. Expression of rat heavy chain ferritin cDNA resulted in increased rates of folate turnover in cultured Chinese hamster ovary cells and human mammary carcinoma cells and reduced intracellular folate concentrations in Chinese hamster ovary cells. These results indicate that ferritin catalyzes folate turnover in vitro and in vivoand may be an important factor in regulating intracellular folate concentrations. tetrahydrofolate p-aminobenzoylglutamate 4-morpholineethanesulfonic acid minimal essential medium polyacrylamide gel electrophoresis heavy chain ferritin light chain ferritin Chinese hamster ovary phosphate-buffered saline 2-(cyclohexylamino)ethanesulfonic acid Tetrahydrofolate (THF)1is a metabolic cofactor that accepts and donates single carbon units and is a source of reducing equivalents for the thymidylate synthase reaction (1Shane B. Vitam. Horm. 1989; 45: 273-335Google Scholar, 2Appling D.R. FASEB J. 1991; 5: 2645-2651Crossref PubMed Scopus (304) Google Scholar). Chemically, THF consists of a quinazoline ring that is bridged to a p-aminobenzoylglutamate (pABG) moiety through the C-9 methylene group (see Scheme FS1). In cells, THF exists in several chemically modified forms. The quinazoline ring can be oxidized to yield dihydrofolate, the product of the thymidylate synthase reaction, and the N-5 and N-10 of the cofactor are modified with single carbon units at the oxidation state of methanol (5-methyl-THF), formaldehyde (5,10-methylene-THF), and formate (10-formyl-THF, 5-formyl-THF, and 5,10-methenyl-THF). Each derivative serves a particular metabolic function in cytoplasmic folate metabolism. 5-Methyl-THF is required to remethylate homocysteine to methionine, whereas 5,10-methylene-THF is required to convert dUMP to dTMP. 10-Formyl-THF supplies C-2 and C-8 for purine ring biosynthesis. 5-Formyl-THF does not serve as a cofactor for folate-dependent anabolic reactions, but is an effective inhibitor of several folate-dependent reactions (3Stover P. Schirch V. Trends Biochem. Sci. 1993; 18: 102-106Abstract Full Text PDF PubMed Scopus (112) Google Scholar, 4Stover P. Schirch V. J. Biol. Chem. 1991; 266: 1543-1550Abstract Full Text PDF PubMed Google Scholar). THF is unstable in vitro and readily undergoes oxidative degradation. Solutions of THF can be stabilized in vitro by the addition of reduced thiols or antioxidants, including ascorbate. Oxidation can occur by at least two distinct mechanisms that are essentially irreversible. The quinazoline ring can be sequentially oxidized to dihydrofolate and then to folic acid through a quinonoid dihydrofolate intermediate (5Reed L.S. Archer M.C. J. Agric. Food Chem. 1980; 28: 801-805Crossref Scopus (54) Google Scholar, 6Chippel D. Scrimgeour K.G. Can. J. Biochem. 1970; 48: 999-1009Crossref PubMed Scopus (51) Google Scholar). This mechanism is also shared by tetrahydropterin oxidation (7Moad G. Luthy C.L. Benkovic S.J. Tetrahedron Lett. 1978; 26: 2271-2274Crossref Scopus (13) Google Scholar, 8Archer M.C. Vonderschmitt D.T. Scrimgeour K.G. Can. J. Biochem. 1972; 50: 1174-1182Crossref PubMed Scopus (44) Google Scholar). The site of oxidation has been proposed to occur through a 4a-carbinolamine intermediate, and chemically stable deazatetrahydropterin 4a adducts have been synthesized (7Moad G. Luthy C.L. Benkovic S.J. Tetrahedron Lett. 1978; 26: 2271-2274Crossref Scopus (13) Google Scholar) and shown to be analogous to intermediates associated with the nonenzymatic oxidation of tetrahydropterins. Similar intermediates are also seen for the phenylalanine hydroxylase-catalyzed oxidation of tetrahydropterins (7Moad G. Luthy C.L. Benkovic S.J. Tetrahedron Lett. 1978; 26: 2271-2274Crossref Scopus (13) Google Scholar, 9Davis M.D. Kaufman S. J. Biol. Chem. 1989; 264: 8585-8596Abstract Full Text PDF PubMed Google Scholar). 2-Mercaptoethanol and other reduced thiols, which serve to protect reduced folates from oxidation (10Zakrzewski S.F. J. Biol. Chem. 1966; 241: 2957-2961Abstract Full Text PDF PubMed Google Scholar), are proposed to protect this site from oxidation by forming transient 4a adducts. This notion is supported by studies demonstrating that lyophilized samples of pure THF contain 1 atom of sulfur/THF molecule (10Zakrzewski S.F. J. Biol. Chem. 1966; 241: 2957-2961Abstract Full Text PDF PubMed Google Scholar). Alternatively, THF or dihydrofolate can undergo an oxidative scission reaction at the C-9–N-10 bond. Electron extraction at N-10 results in the formation of an intermediate N-10 nitrenium ion that rapidly converts to the more stable C-9–N-10 Schiff base. Upon hydrolysis of the Schiff base, the scission products 6-formyltetrahydropterin (or 6-formyldihydropterin) and pABG are generated (5Reed L.S. Archer M.C. J. Agric. Food Chem. 1980; 28: 801-805Crossref Scopus (54) Google Scholar). One carbon substitution at N-5 or N-10 can alter the reactivity of THF to oxidative degradation (11Maruyama T. Shiota T. Krumdieck C.L. Anal. Biochem. 1978; 84: 277-295Crossref PubMed Scopus (54) Google Scholar, 12Lewis G.P. Rowe P.B. Anal. Biochem. 1979; 93: 91-97Crossref PubMed Scopus (34) Google Scholar). 5-Formyl-THF is the most stable derivative of THF, and its stability has been attributed in part to steric protection of the C-4a oxidation site. Neither the role nor biochemical mechanisms of folate turnover in regulating intracellular folate concentration have been widely investigated. Humans turn over 90% of the 5-formyl-THF catabolic activity was associated with a single fraction, with the exception of hydroxylapatite chromatography. Folic acid, 5-formyl-THF, and pterin-6-carboxylic acid affinity columns were synthesized as described previously (22Maras B. Stover P. Valiante S. Barra D. Schirch V. J. Biol. Chem. 1994; 269: 18429-18433Abstract Full Text PDF PubMed Google Scholar). Isolation and purification of intact mitochondria from fresh rat livers were performed as described previously (22Maras B. Stover P. Valiante S. Barra D. Schirch V. J. Biol. Chem. 1994; 269: 18429-18433Abstract Full Text PDF PubMed Google Scholar). 100 fresh-frozen rat livers were thawed and homogenized in 1 liter of 50 mm potassium phosphate (pH 7.2) containing 5% (w/v) polyethylene glycol 8000. The homogenate was centrifuged, and the pellet was discarded. Polyethylene glycol 8000 was added to the supernatant to a final concentration of 17% (w/v), and the solution was centrifuged. The supernatant did not contain any 5-formyl-THF catabolic activity and was discarded. The pellet was dissolved in 500 ml of 50 mm potassium phosphate (pH 7.5), and ammonium sulfate was added to a final concentration of 30%. After centrifugation, the supernatant was discarded. The pellet was resuspended in 500 mm potassium phosphate (pH 7.5). Only this fraction contained 5-formyl-THF cleavage activity. The protein solution from Step 1 was directly applied to a 10 × 10-cm phenyl-Sepharose matrix equilibrated with 500 mm potassium phosphate (pH 7.2). The column was washed with 500 mmpotassium phosphate (pH 7.5) until the A 280 of the eluant was <0.1. The flow-through and wash fractions did not contain detectable 5-formyl-THF catabolic activity. The enzyme activity was eluted from the matrix with 5 mm potassium phosphate (pH 7.2). The activity eluted as a sharp red band. Active fractions were pooled and precipitated with ammonium sulfate (50% saturation). Following centrifugation, the protein pellet was resuspended in 500 mm potassium phosphate (pH 7.2). The protein solution from Step 2 was applied to a 5.5 × 8-cm Butyl-Sepharose column. The column was washed with 500 mm potassium phosphate (pH 7.5) until the A 280 of the eluant was <0.2. The enzyme activity was eluted with a linear gradient; buffer A (200 ml) was 500 mm potassium phosphate (pH 7.2), and buffer B (200 ml) was 5 mm potassium phosphate (pH 7.2). The activity eluted midway through the gradient as a red band. Active fractions were pooled (∼100 ml), precipitated with ammonium sulfate (50% saturation), and dialyzed overnight against 5 mm Tris-Cl (pH 7.5). The dialyzed protein solution from Step 3 was applied to a 5 × 10-cm hydroxylapatite column equilibrated with 5 mm Tris-Cl (pH 7.5). The enzyme activity was eluted with a linear gradient; buffer A (100 ml) was 5 mm Tris-Cl (pH 7.5), and buffer B (100 ml) was 200 mm potassium phosphate (pH 7.2). The most active fraction (see Fig. 2) eluted midway through the gradient as a red band. This fraction was diluted with water to give a final potassium phosphate concentration of 10 mm. The active protein fraction from Step 4 was applied to a 2.5 × 5-cm DEAE-Sephacel column equilibrated with 10 mm potassium phosphate (pH 7.2). The protein was eluted with a linear gradient; buffer A (200 ml) was 5 mmpotassium phosphate (pH 7.2), and buffer B (200 ml) was 200 mm potassium phosphate (pH 7.2). The activity eluted as a red band midway through the gradient. Active fractions were pooled (∼100 ml), precipitated with ammonium sulfate (50% saturation), and dialyzed overnight against 5 mm potassium phosphate (pH 7.2). The dialyzed protein from Step 5 was applied to a 1.5 × 4-cm pterin affinity column pre-equilibrated with 5 mm potassium phosphate (pH 7.2) (buffer A). The column was washed with buffer A until theA 280 was 95%, demonstrating that the catabolism of 5-formyl-THF is enzyme-catalyzed and is not due to small molecule oxidants present in liver. The 5-formyl-THF catabolic activity was purified to homogeneity as described under "Experimental Procedures" (TableI). The activity had been purified by this method >10 times starting from 100 fresh-frozen rat livers. The purification could be completed in 4 days and required the use of six column matrices. Throughout the purification, the 5-formyl-THF catabolic activity was associated with a single, red-colored fraction for all precipitation and chromatography steps, with the exception of the hydroxylapatite column. Fig.1 displays the three major protein fractions that eluted from the hydroxylapatite column (as determined by the A 280), and all three fractions contained a chromophore as demonstrated by the A 310. Each fraction was found to contain high l