F.M. Huennekens

Activation of methionine synthase: Further characterization of the flavoprotein system

Abstract Two homogeneous flavoproteins (R and F components) which, in conjunction with catalytic amounts of NADPH and adenosylmethionine, comprise an efficient system for activation of the B 12 -containing methionine synthase (M component) from Escherichia coli K-12, have been characterized with respect to oxidation-reduction properties and participation in the activation process. The flavin (FAD) of R component is reduced to FADH 2 by NADPH. Reduced R, in turn, reduces the flavin (FMN) of F component to a blue semiquinone (FMNH ·). Reduction potentials (at pH 7.0) for R and F are −0.30 and −0.29 V, respectively. Various other compounds such as ferricyanide, 2,6-dichlorophenol-indophenol, menadione, and -cytochrome c can also serve as electron acceptors for reduced R, but only F can efficiently mediate the NADPH- and R-dependent activation of M component. Activation is assumed, therefore, to involve the sequence: NADPH → R → F → M. During operation of the complete system, the amount of NADPH consumed is less than 2% of the amount of methionine synthesized.

Preparation and properties of crystalline 5-methyl tetrahydrofolate and related compounds

Abstract 5-Methyl tetrahydrofolate (I) has been prepared by the methods of Sakami and Ukstins and Keresztesy and Donaldson (i.e., admixing HCHO with tetrahydrofolate followed by reduction with KBH 4 ), purified by chromatography, and crystallized as both the barium salt and the free acid. Oxidation of (I) with H 2 O 2 in the presence of peroxidase yielded a dihydro compound, presumably 5-methyl-5,6-dihydrofolate (II); reduction of (II) with borohydride or mercaptoethanol regenerated (I). 10-Methyl folate (III) was prepared by alkaline hydrolysis of 4-amino-10-methyl folate (Methotrexate); reduction of (III) with hydrosulfite yielded 10-methyl-7,8-dihydrofolate (IV) as the crystalline acid, while hydrogenation of (III) over PtO 2 produced the oxygen-labile, amorphous 10-methyl-5,6,7,8-tetrahydrofolate (V). Compounds I–V were further characterized by elemental analyses, melting points, absorption spectra at pH 1, 7, and 13, and R f values.

Enzymatic conversion of vitamin B12a to adenosyl-B12: Evidence for the existence of two separate reducing systems

Abstract The reduction of vitamin B 12 a , (Co III ) to B 12 s (Co I ) appears to be carried out by two separate enzyme systems in extracts of Clostridium tetanomorphum . The reduction of B 12 a to B 12 r (Co II ) occurs at a relatively rapid rate and is catalyzed by a DPNH-dependent flavoprotein (B 12 a reductase) that utilizes PAD better than FMN. Trapping experiments suggest that B 12 a is not an intermediate in this reaction. The reduction of B 12 r to B 12 s , a reaction characterized by an unfavorable equilibrium, requires a second DPNH-dependent flavoprotein (B 12 r reductase) that is stimulated equally well by FAD or FMN. In addition to their different responses to added flavins, these systems are also distinguished by other characteristics: ( a ) B 12 a reductase is considerably more labile than the B 12 r reductase; and ( b ) Dithioerythritol can replace DPNH as the reductant for the B 12 r reductase but not for the B 12 a reductase.

Dihydrofolate Reductases: Structural and Mechanistic Aspects*

In cancer chemotherapy the advent of each clinically useful agent has been followed by a search for the molecular receptor or “target” for that drug. In the case of the folate antagonists such as aminopterin or amethopterin (Methotrexate) , which have been used extensively in the treatment of leukemia, lymphoma, and choriocarcinoma,’ the target is presumed to be dihydrofolate reductase, an enzyme whose primary function is catalysis of reaction ( 1 ) , the structural details of which are presented in FIGURE 1.

Binding properties of the 5-methyltetrahydrofolate/methotrexate transport system in L1210 cells☆

Abstract A binding component with a high affinity for 5-methyltetrahydrofolate ( K D = 0.11μ m ) is present on the external surface of L1210 cells. The amount of binder (1 pmol/mg protein) corresponds to 8 × 10 4 sites per cell. The participation of this component in the high-affinity 5-methyltetrahydrofolate/methotrexate transport system is supported by similarities in the K D values for 5-methyltetrahydrofolate and methotrexate binding and the K t values of these compounds for transport. Relative affinities for other folate substrates (aminopterin, 5-formyltetrahydrofolate, and folate) and various competitive inhibitors (thiamine pyrophosphate, ADP, AMP, arsenate, and phosphate) are also similar for both the binding component and the transport system. The measured binding activity does not represent low-temperature transport of substrate into cells, since it is readily saturable with time and is eliminated by either washing the cells with buffer or by the addition of excess unlabeled substrate.

Transport of vitamin B12 into mouse leukemia cells

Abstract Transport of [ 57 Co]cyanocobalamin (vitamin B 12 ) into L1210 murine leukemia cells, mediated by transcobalamin-II, is a biphasic process. The primary step, which occurs very rapidly (within the first minute), appears to involve binding of the B 12 -transcobalamin complex to the external membrane of the cells; at 37 °C and pH 7.3, apparent K m and V values are 180 pM and 5.9 pmol/min/10 9 cells, respectively. This step is relatively insensitive to temperature, has an apparent p K a of 6.0, and is inhibited by EDTA; Ca 2+ or Mg 2+ can reverse the inhibition. The slower secondary step appears to involve translocation of the vitamin into the cell; V for this step is 0.4 pmol/min/10 9 cells. Temperature and pH optima are 30 ° and 6.5−7.0, respectively. This step is stimulated by glucose and inhibited by cyanide, arsenite, arsenate, p -chloromercuriphenylsulfonate and dinitrophenol. Labeled B 12 in an amount corresponding to that taken up in the initial step can be released by: (a) resuspending the cells in a B 12 -free medium; (b) addition of unlabeled B 12 -transcobalamin-II; and (c) addition of EDTA. The material released is chromatographically and functionally identical with B 12 -transcobalamin-II.

Interconversion of the multiple forms of dihydrofolate reductase from amethopterin-resistant Lactobacillus casei

Abstract Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei can be separated into two principal forms (I and II) by electrophoresis on polyacrylamide or by chromatography on ion-exchangers. Form (II) (the component that migrates more rapidly toward the positive electrode during electrophoresis at pH 8.5) contains approximately 1 mole of TPNH per mole of protein (MW = 15,000), as judged by spectral characteristics (absorbance maxima at 274 and 340 mμ and fluorescence maximum at 445 mμ) of the intact protein and by analysis (thin-layer chromatography and coenzymatic activity with yeast glutathione reductase) of the nucleotide released by heat denaturation of the protein. Treatment of (II) with 7,8-dihydrofolate results in oxidation of the bound TPNH, release of the resultant TPN, and creation of form (I). (II) can be resynthesized by incubation of (I) with TPNH at neutral pH.

Folate antagonists covalently linked to carbohydrates: Synthesis, properties, and use in the purification of dihydrofolate reductases

Abstract The folate antagonists, aminopterin and amethopterin, have been linked covalently via their carboxyl groups to various high molecular-weight water-soluble (starch, dextrans) and water-insoluble (cellulose, agarose) carbohydrates. The amount of folate compound incorporated into these polymers was determined by absorbance measurements and by radioactivity when 3H-labeled precursors were used. Optimal yields (ca. 150 mg/g) were obtained by using carbodiimides to effect amide linkages between the folate compounds and aminoalkyl derivatives of the carbohydrates. The soluble antagonist-carbohydrate complexes were characterized with respect to absorbance spectra, molecular weight, and ability to inhibit dihydrofolate reductase. Folate antagonists bound to carbohydrates retained an appreciable affinity for dihydrofolate reductases. This property has provided the basis for two different procedures for purification of these enzymes: (1) complex formation with amethopterin-aminoethyl-soluble starch and isolation of this complex via Sephadex G-100; and (2) affinity chromatography on amethopterin-aminoalkyl-Sepharose. Both methods were facilitated by the ability of dihydrofolate reductases to bind tightly to the antagonist at acidic pH values and the tendency for such complexes to dissociate at alkaline pH values, especially in the presence of dihydrofolate or folate.

THYMIDYLATE SYNTHETASE AND ITS RELATIONSHIP TO DIHYDROFOLATE REDUCTASE

Thymidylate (dTMP) and its triphosphate (dTTP) occupy a somewhat unique position in DNA synthesis. As outlined in FIGURE 1, three of the substrates (dATP, dGTP and dCTP) for DNA polymerase are obtained by direct reduction of the corresponding ribonucleotides, but dTTP is synthesized by a more circuitous route: (1) UTP is reduced (again via ribonucleotide reductase) to dUTP; ( 2 ) dUTP is dephosphorylated to dUMP; ( 3 ) dUMP is converted (via thymidylate synthetase) to dTMP; and (4) dTMP is phosphorylated to dTTP. dTMP can also arise from the phosphorylation of thymidine (dT), but this reaction is probably used only to salvage the previously-formed methylated pyrimidine. Because of its singularity, the methylation reaction (step 3 , above) thus becomes an attractive target for cancer chemotherapy. The structural features of this important reaction are seen in more detail in FIGURE 2. In the presence of the enzyme, 5,1 O-methylene tetrahydrofolate and deoxyuridylate react to form 7,s-dihydrofolate and deoxyuridylate. Since the level of the folate coenzyme is limited within the cell, the continuous synthesis of deoxyuridylate requires that dihydrofolate, resulting from the primary reaction, be reduced to tetrahydrofolate (via the TPNH-dependent dihydrofolate reductase) and that the latter be reconverted (via the addition of the appropriate one-carbon unit) to 5,1 O-methylene tetrahydrofolate. These three reactions, operating in sequence (cf. FIGURE l ) , comprise a thymidylate synthesis cycle.

Interrelationship of adenosyl methionine and methyl-B12 in the biosynthesis of methionine☆

Abstract Methionine synthetase, an enzyme that catalyzes the terminal reaction in the biosynthesis of methionine (5-methyl tetrahydrofolate + homocysteine → tetrahydrofolate + methionine), has been partially purified from pig liver, beef liver, and Escherichia coli . Depending upon the source and the degree of purification, the enzyme shows a requirement for adenosyl methionine, for methyl-B 12 (or methyl cobinamide), or for both of these cofactors. Present evidence supports the assumption that methyl-B 12 (or some compound derived from methyl-B 12 ) is the actual cofactor for the apoenzyme form of methionine synthetase and that adenosyl methionine serves to regenerate methyl-B 12 , which is slowly degraded during its function as the coenzyme, via the reaction (adenosyl methionine + B 12s → methyl-B 12 + adenosyl homocysteine).