Dihydrofolate reductases of Escherichia coli and bacteriophage T4. Spectrofluorometric study

Erickson, John S.; Mathews, Christopher K.

doi:10.1021/bi00727a002

Cited by 36 publications

(16 citation statements)

References 24 publications

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“…The formation of binary complexes between the enzyme and a variety of substrates and inhibitors can Table 5 is comparable with the values found for the enzyme from E. coli (2.4 x 106M-1, Erickson & Mathews, 1973; 9 x 105 M-1, Greenfield et al, 1972), T4 phage (2.0x 106M-1; Erickson & Mathews, 1973) and L1210 lymphoma (5.Ox 106M-1; Perkins & Bertino, 1966). NADPH 1976 appears to bind appreciably more tightly to the L. casei enzyme than to these other enzymes, for which binding constants in the range 1.3 x 106-2 x 107 M-1 were reported.…”

Section: Binding Ofsubstrates and Inhibitorssupporting

confidence: 83%

“…In addition, the two enzymes differ in their activity towards folate (see below). It seems likely that these differences can be attributed to the different strains of bacteria used; similar differences have been noted for the dihydrofolate reductases from different strains of E. coli (Mathews & Sutherland, 1965;Burchall & Hitchings, 1965;Burchall & Chan, 1969;Poe et al, 1972;Erickson & Mathews, 1973) and S. faecalis (see, for example, Albrecht & Hutchinson, 1969;D'Souza et al, 1972).…”

Section: Enzyme Assaymentioning

confidence: 69%

“…Thus the E. coli enzyme, for example, shows only a decrease in activity on increasing the salt concentration above 50mM Erickson & Mathews, 1973), although Nixon & Blakley (1968) reported a slight activation by 0.2M-KCI for the enzyme from S. faecium. Dihydrofolate reductase from mammalian sources is also activated by urea (2-AM) and by thiol reagents, notably mercurials (Blakley, 1969;Perkins & Bertino, 1965;Kaufman, 1968;Rowe & Russel, 1973 Kaufman (1968) reported that activation by urea and related compounds was much less marked with the substrate pairs NADPH/folate and NADH/ H2folate.…”

Section: Kinetic Propertiesmentioning

confidence: 97%

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Large-scale purification and characterization of dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei

Dann¹,

Ostler²,

Bjur³

et al. 1976

Biochemical Journal

150

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Dihydrofolate reductase has been purified from a methotrexate-resistant strain of Lactobacillus casei NCB 6375. By careful attention to growth conditions, up to 2.5g of enzyme is obtained from a 400 litre culture. The purification procedure, involving polyethyleneimine treatment, DEAE-cellulose chromatography and affinity chromatography on methotrexate-aminohexyl-Sepharose, operates on the gram scale, with overall yields of 50-60%. Elution of the affinity column by reverse (upward) flow was used, as it led to recovery of the enzyme in a much smaller volume. The enzyme obtained appears to be more than 98% pure, as judged by gel electrophoresis, isoelectric focusing, and gel filtration. It has a mol.wt. ofapprox. 17900 and a turnover number of4s-(5OmM-triethanolamine/400mM-KCI, pH7.2, 25°C) with dihydrofolate and NADPH as substrates.The turnover number for folate is 0.02s-1. Michaelis constants for a variety of substrates have been measured by using a new fluorimetric assay (0.361uM-dihydrofolate; 0.78 uM-NADPH), and binding constants determined by using the quenching of protein fluorescence (dihydrofolate, 2.25 x 106M-1; NADPH, >108M-1). The pH/activity profile shows a single maximum at pH 7.3; at this pH, marked activation by 0.5M-NaCl is observed.Dihydrofolate reductase (tetrahydrofolate-NADP+ oxidoreductase, EC 1.5.1.3) is responsible for maintaining the intracellular pool of tetrahydrofolate by reducing dihydrofolate (arising either by biosynthesis de novo or by the action of thymidylate synthetase on 5,10-methylenetetrahydrofolate) to tetrahydrofolate (Blakley, 1969). This enzyme is of considerable pharmacological interest as the site of action of a group of powerful chemotherapeutic agents, the 'anti-folates', which includes methotrexate, trimethoprim and pyrimethamine (Blakley, 1969;Baker, 1967).We are undertaking a detailed study of the binding of substrates, coenzymes and inhibitors to the dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei, principally by highresolution n.m.r. (nuclear-magnetic-resonance) spec-* Present address:

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Section: Binding Ofsubstrates and Inhibitorssupporting

confidence: 83%

Section: Enzyme Assaymentioning

confidence: 69%

Section: Kinetic Propertiesmentioning

confidence: 97%

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Large-scale purification and characterization of dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei

Dann¹,

Ostler²,

Bjur³

et al. 1976

Biochemical Journal

150

View full text Add to dashboard Cite

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“…This value of approx. 21000 is in contrast with values of 17000 for DHFR from an MTX-resistant strain of E. coli (Poe et al, 1972) and 17800 for DHFR from a TMP-resistant strain of E. coli (Baccanari et al, 1975) among others (Mathews & Sutherland, 1965;Burchall & Hitchings, 1965;Erickson & Mathews, 1973). It seems that the differences can be ascribed to the different bacterial strains used, similar differences having been noted for DHFR from different strains of L.…”

Section: Resultsmentioning

confidence: 77%

Interactions between inhibitors of dihydrofolate reductase

Bowden

Hall

Birdsall³

et al. 1989

Biochemical Journal

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The binding of substrates and inhibitors to dihydrofolate reductase was studied by steady-state kinetics and high-field 1H-n.m.r. spectroscopy. A series of 5-substituted 2,4-diaminopyrimidines were examined and were found to be 'tightly binding' inhibitors of the enzyme (Ki less than 10(-9) M). Studies on the binding of 4-substituted benzenesulphonamides and benzenesulphonic acids also established the existence of a 'sulphonamide-binding site' on the enzyme. Subsequent n.m.r. experiments showed that there are two binding sites for the sulphonamides on the enzyme, one of which overlaps the coenzyme (NADPH) adenine-ring-binding site. An examination of the pH-dependence of the binding of sulphonamides to the enzyme indicated the influence of an ionizable group on the enzyme that was not directly involved in the sulphonamide binding. The change in pKa value from 6.7 to 7.2 observed on sulphonamide binding suggests the involvement of a histidine residue, which could be histidine-28.

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“…Binding of a cation in the active site of acetylcholine esterase appears to be responsible for a pK a shift that is observed experimentally but not in electrostatic calculations (Wlodek et al, 1995). DHFR is known to be inhibited by ions (Poe et al, 1972;Erickson & Mathews, 1973;Baccanari et al, 1975), and the buffer used in the NMR studies contained 100 mM potassium phosphate. However, given that the pK a of MTX appears to be shifted even when MTX and the cofactor NADP are bound, this phenomenon seems less likely in this case.…”

Section: Inhibitor Binding: Salt-bridge or Neutral Polar Interaction?mentioning

confidence: 99%

Consideration of the pH-dependent inhibition of dihydrofolate reductase by methotrexate 1 1Edited by B. Honig

Cannon

Garrison

Benkovic

1997

Journal of Molecular Biology

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Dihydrofolate reductases of Escherichia coli and bacteriophage T4. Spectrofluorometric study

Cited by 36 publications

References 24 publications

Large-scale purification and characterization of dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei

Large-scale purification and characterization of dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei

Interactions between inhibitors of dihydrofolate reductase

Consideration of the pH-dependent inhibition of dihydrofolate reductase by methotrexate 1 1Edited by B. Honig

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