Dihydrofolate Reductases: Structural and Mechanistic Aspects

Huennekens, F.M.; Dunlap, R. Bruce; Freisheim, James H.; Gundersen, L. E.; Harding, Nigel G. L.; Levison, Stuart A.; Mell, Galen P.

doi:10.1111/j.1749-6632.1971.tb31129.x

Cited by 11 publications

(12 citation statements)

References 28 publications

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“…Evaporation under reduced pressure left a red glass, which was chromatographed on silica gel (300 g) with 3:7 EtOAc-benzene as the eluent. The main fraction yielded 5a (tic on silica gel, R¡ ~0.3) as a yellow oil (10.5 g, 45%): ir (CHC13) 2.90 (NH2), 4.50 (CN), 5.80…”

Section: Experimental Section § A-[[p-(a-p-toiuenesulfonyl)methylamin...mentioning

confidence: 99%

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Methotrexate analogs. 3. Synthesis and biological properties of some side-chain altered analogs

Chaykovsky¹,

Rosowsky²,

Papathanasopoulos³

et al. 1974

J. Med. Chem.

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Section: Experimental Section § A-[[p-(a-p-toiuenesulfonyl)methylamin...mentioning

confidence: 99%

“…[ [ (2,4 -Di amino-6-pteridinyl )methyl]methylamino]benzoylj-2-aminopentane (6c). Condensation of 5c with excess guanidine in refluxing EtOH for 1 hr, cooling, and filtration of the precipitated yellow solid gave 6c (80%): mp 264-266°dec; uv X max (EtOH) 261 nm (< 25,000), 291 (23,800), 375 (7830).…”

Section: N-[p -mentioning

confidence: 99%

Methotrexate analogs. 3. Synthesis and biological properties of some side-chain altered analogs

Chaykovsky¹,

Rosowsky²,

Papathanasopoulos³

et al. 1974

J. Med. Chem.

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“…The dismutation activity of DHFR is analogous to its transhydrogenase activity reported some time ago by Huennekens et al (1971). In the latter case the enzyme was reported to catalyze the reduction of the acetylpyridine analogue of NADP"1" by NADPH.…”

Section: Resultsmentioning

confidence: 59%

“…The presence of bound nucleotide in preparations of purified DHFR that was disclosed when the dismutation reaction was investigated is surprising and indeed paradoxical in view of the relatively high dissociation constants determined by fluorescence titration for complexes of NADPH and especially for NADP"1" (Table III). However, isolation of such relatively stable complexes has been reported in the past for DHFR from L. casei (Huennekens et al, 1971; Gundersen et al, 1972), from chicken liver (Huennekens et a., , 1971Mell et al, 1968), and from methotrexate-resistant L1210 murine lymphoma cells (Huennekens et al, 1971;Harding et al, 1970;Neef & Huennekens, 1975). How the relative stability of these complexes, as exemplified by their survival during enzyme purification and during procedures such as gel electrophoresis, can be reconciled with the measured dissociation constants is still unclear.…”

Section: Resultsmentioning

confidence: 99%

Dismutation of dihydrofolate by dihydrofolate reductase

Blakley¹,

Cocco²

1984

Biochemistry

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Degradation of 7,8-dihydrofolate (H2folate) in the presence of dihydrofolate reductase (DHFR) has been shown due not to an oxygenase activity of the reductase as previously reported but to dismutation of H2folate to folate and 5,6,7,8-tetrahydrofolate (H4folate). The reaction can be followed spectrophotometrically or by analysis of the reaction mixture by high-performance liquid chromatography (HPLC). The products have also been isolated and characterized. Oxygen uptake during the reaction is much less than stoichiometric with H2folate disappearance and is attributed to autoxidation of the H4folate formed. The dismutation activity is a property of highly purified Streptococcus faecium DHFR isoenzyme 2 (but not isoenzyme 1) and of Lactobacillus casei DHFR, but not of bovine liver DHFR. The activity is dependent on tightly bound NADP+ and/or NADPH. Removal of the nucleotide results in loss of dismutation activity, which is restored by adding NADP+ or NADPH. Maximum activity is obtained when approximately 1 mol equiv of nucleotide is added per mol of DHFR. It is proposed that in the dismutation reaction bound NADP(H) is alternately reduced and oxidized by incoming molecules of H2folate with release of folate and H4folate, respectively. The relatively slow rate of folate formation presumably limits the rate of the overall reaction. The equilibrium constant for the dismutation reaction is 19.4 +/- 7.4 at 22 degrees C and pH 7.0. Calculation of standard oxidation-reduction potentials at pH 7 gave values of -0.230 V for the H2folate/H4 folate pair and -0.268 V for the folate/H2folate pair. The mechanism by which NADP+ is retained by the enzyme from some sources during purification procedures is unclear.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…52 mU aufgetragen. Diese reagiert mit dem Tetrazoliumsalz MTT unter Bildung eines sehr schlecht wasserlSslichen, lichtechten violett-blauen Formazans(13,14), das somit zum Indikator der FH2-Reduktase wird. Bei einer Laufdauer von 75-120 Minuten konnte jeweils eine genUgend gute Trennung der Enzymbanden erreicht werden.…”

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Hochspannungselektrophorese der Dihydrofolatreduktase aus Escherichia coli W 3110

Schalhorn

Wilmanns

1977

Res. Exp. Med.

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A method for the high-voltage electrophoresis of dihydrofolate reductase from Escherichia coli W 3110 is described. Dihydrofolate reductase catalyses the reduction of dihydrofolic acid to tetrahydrofolic acid. By addition of a tetrazolium salt, tetrahydrofolic acid reacts by formation of a violet water insoluble formazane which is an indicator for the enzyme. Besides several unspecific bands, two isoenzymes of the dihydrofolate reductase from Escherichia coli W 3110 are found which are specificially inhibited by the folate antagonists methotrexate and trimethoprime in a concentration of 0, 1muM, 1 muM respectively.

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Dihydrofolate Reductases: Structural and Mechanistic Aspects

Cited by 11 publications

References 28 publications

Methotrexate analogs. 3. Synthesis and biological properties of some side-chain altered analogs

Methotrexate analogs. 3. Synthesis and biological properties of some side-chain altered analogs

Dismutation of dihydrofolate by dihydrofolate reductase

Hochspannungselektrophorese der Dihydrofolatreduktase aus Escherichia coli W 3110

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