Characterization of the Dihydropterin Reductase Activity of pig Liver Methylenetetrahydrofolate Reductase*

Pig liver methylenetetrahydrofolate reductase catalyzes the reduction of quinonoid dihydro- pterins in vitro. Either NADPH or methyltetrahydrofolate can serve as the electron donor. Methylenetetrahydrofolate reductase can also support phenylalanine hydroxylation in vitro by regeneration of the tetrahydropterin cofactor. These results lend support to the proposal that reduction of methylenetetrahydrofolate proceeds by tautomerization of the 5-iminium cation to form quinonoid 5-methyldihydrofolate, which is then reduced to methyltetrahydrofolate (Mat- thews, R. G., and Haywood, B. J. (1979) Biochemistry 18,4845-4851). Under V,, conditions, the turnover numbers for the NADPH-linked reductions of the quinonoid forms of 6,?-dimethyldi- hydropterin, dihydrobiopterin, and dihydrofolate are all about the same as that for the reduction of methylenetetrahydrofolate. The K,,, values for racemic mixtures of the same quinonoid accep-tors are 40, 30, and 20 p ~ , respectively, while the K,,, for (GR,S)methylenetetrahydrofolate is 20 p~ at pH 7.2 in phosphate buffer. The reduction of quinonoid dihydropterins is inhibited by adenosylmethionine and dihydropteroylhexaglutamate, which are known to modulate methylenetetrahydrofolate reductase activity.

* This work has been supported in part by Research Grant GM 24908 from the National Institute of General Medical Sciences, National Institutes of Health, United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertzsement" in accordance with 18  DMPH2, HJiopterin, and Hpfolate are comparable to that for CHP-H,folate.

MATERIALS AND METHODS
CH2-H4folate reductase was purified from pig liver as previously described (3) except that the Affgel-Blue column was washed with 1 mM NADH (2 d / m l column bed volume) prior to elution of the reductase with 2 M NaCl. For certain preparations, the enzyme was concentrated by ultrafiltration and chromatographed on Sephacryl S-200 equilibrated with 0.025 M phosphate buffer, pH 7.2, 0.15 mM in EDTA, and 0.5 M in NaCI.
H4biopterin was prepared by catalytic hydrogenation of biopterin (4) which was obtained from Regis Chemicals; DMPH? was a gift from Professor Stephen Benkovic or was obtained from Sigma; and H,PtGlu6 was prepared as previously described (5). In experiments where [I4C]phenylalanine was converted to ['4C]tyrosine, the amino acids were separated by chromatography on sheets of Eastman precoated cellulose (No. 6064) as described by Kaufman (6). Details of assay procedures are included in the table legends.
Electrophoresis was performed according to Davis (7) and Ornstein (8) using a Hoefer slab gel electrophoresis apparatus. Protein bands were stained with Coomassie blue and activity was localized by formazan deposition in a solution containing 100 PM NADPH, 100 PM quinonoid DMPH? (prepared immediately before use by oxidation with bromine), 1.2 mM MTT, 0.3 IIUU EDTA, and 2 pM FAD in 0.05 M Tris/CI buffer, pH 7.2 (9, IO).
An apparent molecular weight based on the Stokes radius W~S obtained by chromatography of CH2-H4folate reductase on a Sephacryl S-200 column which had been calibrated with ribonuclease, thymotrypsinogen A, ovalbumin, bovine serum albumin, pig heart lipoamide dehydrogenase, and human y-globulin.

RESULTS
CH,-H,folate reductase, purified by chromatography on Sephacryl S-200, had a specific activity range of 1.2 to 1.8 pmol of CHs-Hrfolate oxidized min" mg" at 37°C. These enzyme preparations are purified 4000-to 6000-fold from liver homogenate, and show the absorbance characteristics of a typical flavoprotein (maxima at 450 and 380 nm, with a prominent shoulder at 480 nm). The flavin can be reduced by either NADPH or CH,,-H,folate. The enzyme preparations were heterogeneous on slab gel electrophoresis, but only one band was stained with either NADPH/MTT or NADPH/ MTT/DMPH,. The protein band associated with activity comprised about 20% of the total protein on the gels. The purified enzyme had an M, of 180,000.
Enzyme which is purified by chromatography on Affigel-Blue, but not chromatographed on Sephacryl S-200, has a specific activity range of 0.4 to 0.6 pmol of CH:%-H4folate oxidized min" mg".
The data in Table I  '' All assays were performed at 25OC in 50 mM Tris/Cl buffer, pH 7.2, 0.3 rnM in EDTA and 2 p~ in FAD. Quinonoid DMPHL was generated immediately before use by oxidation with a stoichiometric quantity of bromine (11). Each assay contained 250 nmol of ["Cmethy[lH,folate (ZOO0 dprn nmol") in 0.5 ml. CH:l-H,folate oxidation was measured by addition of dirnedone at the end of the reaction, heating at 100°C for 2 rnin, extraction of the dimedone.formaldehyde complex into toluene, and counting in Econofluor scintillation fluid. An aliquot of enzyme containing 0.12 mg of protein (specific activity, 0.4 pmol min" rng" at 37°C) was added to each assay.
in the activity of DMPH, as an electron acceptor after 60 min suggests that only the quinonoid tautomer is a substrate for reduction. Air oxidation is also known to convert H4pterins to quinonoid HPpterins (12). In the presence of air, but not under nitrogen, both DMPH4 and H,folate form products which serve as electron acceptors. Under the conditions of these experiments, the half-life of DMPH4 in air-saturated buffer is about 15 min.
We have also measured the pyridine nucleotide-dependent reduction of quinonoid Hzpterins, and our data are summarized in Table 11. We have compared the absorbance changes seen during enzymatic reduction of DMPH, generated by two different methods: by using HzOs/peroxidase or by oxidizing DMPH, with stoichiometric aliquots of bromine (11) immediately prior to the assay. In the latter case, the extinction coefficient which should be used to convert measured absorbance changes at 340 nm to a velocity in moles of substrate reduced min" is 13,100 M" cm", which is the sum of the E~~~~ due to NADPH oxidation and the E :~, ,~ due to reduction of quinonoid dihydropterin (6900 M" cm" (4)). When these measurements were performed at DMPH, concentrations from 20 to 150 p~, the same absorbance changes were observed with both methods of forming DMPH,, indicating that we should employ an e340 of 13,100 M" cm" in both cases. The rate of peroxidase-catalyzed oxidation of H4pterins is in the order DMPH, > H4biopterin > Hdfolate, and is greatly inhibited by 100 PM NADPH or NADH, the reduced pyridine nucleotide concentration routinely used in dihydropteridine reductase assays (13,14).
The data in Table I1 demonstrate the rather marked effect of phosphate buffer on VmaX and on the K , for NADH associated with CH~-H,folate reduction. These rather unusual properties of the reductase are also reflected in the reduction of quinonoid DMPH,. These data also indicate that the K,,, values for quinonoid dihydropterin substrates are comparable in magnitude to the K , for CHa-H4folate.
Since CHa-H4folate reductase is inhibited by H2folate and its polyglutamate analogues, with the lowest K, being that for HsPteGluh (4), we have examined the effect of this inhibitor on the reduction of quinonoid DMPH2. HePteGluti is competitive with respect to DMPH2, with a K, of 45 nM in 50 mM phosphate buffer, pH 7.2. Reduction of 150 PM DMPHz is almost completely inhibited by 2 p~ HpPteGlur;. Reduction of quinonoid DMPH, is also inhibited by adenosylmethionine, which is an allosteric inhibitor of CH2-H4folate reductase (2).  Table I11 compares the stimulation of phenylalanine-dependent NADPH oxidation by CH,-H,folate reductase and dihydropteridine reductase. CHn-H4folate reductase can substitute for dihydropteridine reductase in supporting phenylalanine-dependent NADPH oxidation, and its activity, but not that of sheep liver dihydropteridine reductase, is inhibited by adenosylmethionine. The specificity of the coupled assay for pyridine nucleotide has also been examined. In the presence of CH~-H,folate reductase, 150 p~ NADPH and NADH are about equally effective in phosphate buffer, whereas in the presence of sheep liver dihydropteridine reductase, the activity is markedly higher with NADH than with NADPH.
The data in Table IV show the ability of CH2-H4folate reductase to support phenylalanine hydroxylation, as measured by tyrosine formation. Similar results were obtained when the hydroxylation reaction was followed by measure-   1 and 2). This stimulation was due to the nonenzymatic reduction of quinonoid dihydropterins by reduced pyridine nucleotides (16).
The "reductase-dependent" formation of tyrosine is that amount formed in the presence of both NADPH and reductase less the amount formed in the presence of NADPH but in the absence of reductase.
~ ~ " ment of the conversion of labeled phenylalanine to tyrosine. The product of the hydroxylation reaction in the presence of C H~H~f o l a t e reductase and either NADPH or CHn-H4folate was shown to be tyrosine by its co-chromatography with authenLic [,"H]tyrosine. DISCUSSION Since the preparations of CH2-H4folate reductase used for these experiments were not homogeneous, it was important to establish that the quinonoid dihydropterin reductase activity was not due to contaminating NADH-dependent dihydropteridine reductase (EC 1.6.99.10). We have modified our purification procedure to attempt to remove any possible contaminating dihydropteridine reductase. Since dihydropteridine reductase is adsorbed onto columns containing immobilized Cibacron Blue F3GA (the ligand on Affigel-Blue columns) but can be eluted with 10 p~ NADH (16), we rinsed our Affigel-Blue columns with 1 mM NADH prior to elution of CHr-H,folate reductase. No NADH or NADPH-dependent dihydropteridine reductase activity has been detected in the NADH eluate. The M , of dihydropteridine reductase is 42,000 to 50,000 (14,17), whereas that of CH2-H4folate reductase is about 180,000. Thus, chromatography on Sephacryl S-200 should have separated these two enzymes if both were present. Only one peak of activity is seen on chromatography, corresponding to an M , of about 180,000. Slab gel electrophoresis shows only one band staining for activity, and no bands are seen on staining with NADH/MTT/DMPH, in Tris buffer, conditions which should be optimal for staining of dihydropteridine reductase. Thus, we feel that dihydropteridine reductase is not a contaminant of our preparations. The complete inhibition of NADPH-dependent dihydropterin reductase activity by 2 p~ H2PteGlu6, using an enzyme preparation which had not been chromatographed on Sephacryl S-200, also suggests that all the dihydropterin reductase activity is due to CH?-Hjfolate reductase.
Recently, Nakanishi and co-workers (18) have described the isolation of a homogeneous NADPH-dependent dihydropteridine reductase from bovine liver. This enzyme shows almost complete specificity for NADPH in Tris buffer, as does CH,-H.lfolate reductase. However, its M , is about 70,000 and its Kn, for NADPH is less than 1 p~, suggesting that this too is a distinct enzyme showing dihydropteridine reductase activity.
One of our interests in testing CHa-H4folate reductase for its ability to support phenylalanine hydroxylation in citro was to ascertain whether this enzyme might be capable of playing a role in support of H4biopterin-dependent hydroxylation reactions in vivo. The two major tissues where such a role deserves consideration are liver and brain. Liver is the organ where the bulk of phenylalanine hydroxylation occurs. Although there is a large excess of dihydropteridine reductase activity (14) over CH2-H4folate reductase activity' in rat liver, the activity of CH2-H4folate reductase appears to exceed that of phenylalanine hydroxylase (19). Lack of dihydropteridine reductase leads to hyperphenylalaninemia as well as to neurological deterioration (20)(21)(22). It is not certain to what extent, If any, CH2-H4folate reductase is able to support phenylalanine hydroxylation in these patients. The finding that their hyperphenylalaninemia is not invariably severe (23,24) and that some of them can tolerate larger amounts of dietary phenylalanine than can patients with classic phenylketonuria (23) strongly suggests that some phenylalanine hydroxylation occurs and hence, that some reduction of quinonoid ' C. Daubner and K. G. Matthews. unpublished data.
Hzbiopterin must be going on. A portion of this residual phenylalanine hydroxylation may be supported by CH?-H4folate reductase. CH,-H4folate reductase may also be able to support limited rates of tyrosine and tryptophan hydroxylation in brain, particularly in the absence of dihydropteridine reductase. Such a role could account for the finding that a patient with no immunologically or enzymatically detectable dihydropteridine reductase in brain, who had low rates of turnover of dopamine and serotonin in his central nervous system, nevertheless had detectable amounts of both dopamine and serotonin in a biopsy sample of brain (25).
It will also be interesting to determine whether CHr-H,folate reductase plays any role as a dihydropterin reductase in the biosynthesis of dopamine, norepinephrine, and serotonin under normal conditions. Two patients with homocysteinuria due to deficiency of CH2-H,folate reductase have been reported to have lower than normal levels of the metabolites of these neurotransmitters in their cerebrospinal fluids (26). Such findings would be consistent with a role for CH2-H,folate reductase in the synthesis of these neurotransmitters.