Studies on the Inhibition of Dihydrofolate Reductase by the Folate Antagonists

Bertino, Joseph R.; Booth, Barbara A.; Bieber, Allan L.; Cashmore, Arlene R.; Sartorelli, Alan C.

doi:10.1016/s0021-9258(18)51705-1

Cited by 164 publications

(22 citation statements)

References 26 publications

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“…Although the binding constant is based in part on kinetic rather than equilibrium measurements, it is probably within a factor of 2 of the true thermodynamic equilibrium constant. This equilibrium constant is the relevant one for most studies of the inhibition of the enzyme by methotrexate, since the enzyme is commonly assayed in the presence of a large excess of NADPH, and is consistent with earlier upper limits to the K¡ of methotrexate of 10"10-10"u M (Werkheiser, 1961;Bertino et al, 1964;Williams et al, 1973a). Williams et al (1979) have estimated the K¡ of methotrexate for the S.faecium enzyme as 5.8 X KE11 M, and Jackson et al (1976) have reported values ranging from 5.3 X 1(E12 M to 1.35 X '10 M for the reductases from four mammalian cell lines.…”

Section: Discussionsupporting

confidence: 83%

Binding of coenzyme analogs to Lactobacillus casei dihydrofolate reductase: binary and ternary complexes

Birdsall¹,

Burgen²,

Roberts³

1980

Biochemistry

105

102

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The binding, or association, constants of NADP+ NADPH, and a series of structural analogues to dihydrofolate reductase from Lactobacillus casei MTX/R have been determined fluorometrically. Modification of the adenine or nicotinamide rings has little effect on the binding of the oxidized coenzyme, but the thionicotinamide and acetylpyridine analogues of the reduced coenzyme bind much more weakly than NADPH itself. In the presence of the substrate, folate, or the inhibitors methotrxate or trimethoprim, the oxidized coenzymes bind appreciably more tightly to the enzyme. The magnitude of this "cooperativity", which covers a range of 1-37 fold, depends markedly on the structure of both the coenzyme and the substrate or substrate analogue; the nicotinamide ring of the coenzymes is clearly important in these effects. The binding constants of the reduced coenzymes in the presence of methotrexate or trimethoprim were too high to be measured fluorometrically. The dissociation rate constants of the coenzymes from their ternary complexes were therefore measured and compared with the values for the binary complexes reported by Dunn and co-workers [Dunn, S. M. J., Bathchelor, J. G., & King, R. W.(1978) Biochemistry 17, 2356]. The presence of the inhibitors leads to very substantial decreases in the coenzyme dissociation rate constant--by factors of 300-2200. The binding constant of methotrexate in the ternary complex is calculated to be approximately 1.3 X 10(12) M-1. The structural origins of the differences in binding constant and cooperative behavior of the various coenzymes and coenzyme analogues are discussed in the light of information from crystallography and NMR spectroscopy.

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Section: Discussionsupporting

confidence: 83%

Binding of coenzyme analogs to Lactobacillus casei dihydrofolate reductase: binary and ternary complexes

Birdsall¹,

Burgen²,

Roberts³

1980

Biochemistry

105

102

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“…Amethopterin is a potent inhibitor of dihydrofolate reductase. At neutral pH values the affinity of the enzyme for the inhibitor is less than that at more acid pH values; in fact, at pH values of 6 and below, amethopterin is a "stoichiometric" inhibitor of dihydrofolate reductase (Bertino et al, 1964;Werkheiser, 1961). The observation that this inhibitor interacts with the enzyme on an equimolar basis at low pH has led to the use of this agent as an active-site titrant in the determination of molecular weights of purified preparations of the reductase (reviewed by Blakley, 1969, andby Huennekens et al, 1972).…”

Section: Resultsmentioning

confidence: 99%

Circular dichroic studies of the interaction of dihydrofolate reductase with substrates, coenzymes, and inhibitors

D’Souza¹,

Freisheim²

1972

Biochemistry

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d'souza and freisheimvolving the second cysteine residue is suggested by the instability of the inactive disulfide enzyme. Oxidation of the enzyme under aerobic conditions could produce the inactive enzyme, which, in effect, would terminate glycolytic activities. This possible role among others can be studied in yeast cellfree systems, by employing the yeast dehydrogenase which contains only cysteines-149 and-153.References

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“…The results are expressed as mó1 labeled MTX bound/mg protein . DHFR activity was estimated by measuring the decrease in absorbance at 340 nm, according to a method adapted from Bertino et al . (1964) .…”

Section: Dihydrofolate Reductase Binding and Activity Assaysmentioning

confidence: 99%

Dihydrofolate reductase gene amplification-associated shift of differentiation in methotrexate-adapted HT-29 cells.

Lesuffleur

Barbat

Luccioni

et al. 1991

The Journal of Cell Biology

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Abstract. Postconfluent cultures of HT29 cells form a heterogeneous multilayer of which >95% of the cells are undifferentiated . In contrast, when stably adapted to normally lethal concentrations of methotrexate (10~-10-5 M), they form a monolayer of gobletlike cells (Lesuffieur et al ., 1990) which secrete large quantities of mucins and display a discrete brush border with the presence of villin, dipeptidylpeptidase-IV, and carcinoembryonic antigen . When adapted to even higher concentrations of methotrexate (10-4 and

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Studies on the Inhibition of Dihydrofolate Reductase by the Folate Antagonists

Cited by 164 publications

References 26 publications

Binding of coenzyme analogs to Lactobacillus casei dihydrofolate reductase: binary and ternary complexes

Binding of coenzyme analogs to Lactobacillus casei dihydrofolate reductase: binary and ternary complexes

Circular dichroic studies of the interaction of dihydrofolate reductase with substrates, coenzymes, and inhibitors

Dihydrofolate reductase gene amplification-associated shift of differentiation in methotrexate-adapted HT-29 cells.

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