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Literature summary extracted from

  • Volpato, J.P.; Fossati, E.; Pelletier, J.N.
    Increasing methotrexate resistance by combination of active-site mutations in human dihydrofolate reductase (2007), J. Mol. Biol., 373, 599-611.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.5.1.3 expresion in Escherichia coli and CHO cell Homo sapiens

Protein Variants

EC Number Protein Variants Comment Organism
1.5.1.3 F31A/F34V/Q35H methotrexate-resistant mutant, more than 4000fold decrease in ratio kcat/KM Homo sapiens
1.5.1.3 F31G/Q35N methotrexate-resistant mutant, more than 50fold decrease in ratio kcat/KM Homo sapiens
1.5.1.3 F31P methotrexate-resistant mutant, more than 30fold decrease in ratio kcat/KM Homo sapiens
1.5.1.3 F31P/Q35E methotrexate-resistant mutant, more than 200fold decrease in ratio kcat/KM Homo sapiens
1.5.1.3 F31P/Q35H methotrexate-resistant mutant, more than 150fold decrease in ratio kcat/KM Homo sapiens
1.5.1.3 F31R/F34A/Q35N methotrexate-resistant mutant, more than 5000fold decrease in ratio kcat/KM Homo sapiens
1.5.1.3 F31R/F34T/Q35R methotrexate-resistant mutant, more than 7000fold decrease in ratio kcat/KM Homo sapiens
1.5.1.3 F31R/F34T/Q35S methotrexate-resistant mutant, more than 2000fold decrease in ratio kcat/KM Homo sapiens
1.5.1.3 F31R/Q35E methotrexate-resistant mutant, more than 70fold decrease in ratio kcat/KM Homo sapiens
1.5.1.3 F31S/Q35E methotrexate-resistant mutant, more than 450fold decrease in ratio kcat/KM Homo sapiens
1.5.1.3 additional information characterization of ten methotrexate-resistant mutants with different amino acid combinations at residues F31, F34, and Q35. Higher resistance roughly correlates with a greater number of mutations, and an inverse correlation is observed between resistance and catalytic efficiency. Methotrexate-resistant dihydrofolate reductase can protect eucaryotic cells from methotrexate toxicity after transfection into dihydrofolate reductase knock-out cells Homo sapiens

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.5.1.3 methotrexate
-
 Homo sapiens

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.5.1.3 0.000075
-
 7,8-dihydrofolate wild-type, pH 7.6, 23°C Homo sapiens
1.5.1.3 0.00069
-
 7,8-dihydrofolate mutant F31R/Q35E, pH 7.6, 23°C Homo sapiens
1.5.1.3 0.0015
-
 7,8-dihydrofolate mutant F31P, pH 7.6, 23°C Homo sapiens
1.5.1.3 0.0017
-
 7,8-dihydrofolate mutant F31G/Q35N, pH 7.6, 23°C Homo sapiens
1.5.1.3 0.0017
-
 7,8-dihydrofolate mutant F31R/F34T/Q35S, pH 7.6, 23°C Homo sapiens
1.5.1.3 0.002
-
 7,8-dihydrofolate mutant F31R/F34T/Q35R, pH 7.6, 23°C Homo sapiens
1.5.1.3 0.0021
-
 7,8-dihydrofolate mutant F31P/Q35H, pH 7.6, 23°C Homo sapiens
1.5.1.3 0.0022
-
 7,8-dihydrofolate mutant F31R/F34A/Q35N, pH 7.6, 23°C Homo sapiens
1.5.1.3 0.0027
-
 7,8-dihydrofolate mutant F31P/Q35E, pH 7.6, 23°C Homo sapiens
1.5.1.3 0.0033
-
 7,8-dihydrofolate mutant F31S/Q35E, pH 7.6, 23°C Homo sapiens
1.5.1.3 0.0043
-
 7,8-dihydrofolate mutant F31A/F34V/Q35H, pH 7.6, 23°C Homo sapiens

Organism

EC Number Organism UniProt Comment Textmining
1.5.1.3 Homo sapiens P00374
-
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.5.1.3 7,8-dihydrofolate + NADPH + H+
-
 Homo sapiens 5,6,7,8-tetrahydrofolate + NADP+
-
 ?

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.5.1.3 0.5
-
 7,8-dihydrofolate mutant F31R/F34A/Q35N, pH 7.6, 23°C Homo sapiens
1.5.1.3 1
-
 7,8-dihydrofolate mutant F31R/F34T/Q35R, pH 7.6, 23°C Homo sapiens
1.5.1.3 1
-
 7,8-dihydrofolate mutant F31R/F34T/Q35S, pH 7.6, 23°C Homo sapiens
1.5.1.3 1.1
-
 7,8-dihydrofolate mutant F31A/F34V/Q35H, pH 7.6, 23°C Homo sapiens
1.5.1.3 1.3
-
 7,8-dihydrofolate mutant F31R/Q35E, pH 7.6, 23°C Homo sapiens
1.5.1.3 1.7
-
 7,8-dihydrofolate mutant F31P/Q35E, pH 7.6, 23°C Homo sapiens
1.5.1.3 1.7
-
 7,8-dihydrofolate mutant F31P/Q35H, pH 7.6, 23°C Homo sapiens
1.5.1.3 4.3
-
 7,8-dihydrofolate mutant F31G/Q35N, pH 7.6, 23°C Homo sapiens
1.5.1.3 6.4
-
 7,8-dihydrofolate mutant F31P, pH 7.6, 23°C Homo sapiens
1.5.1.3 9.4
-
 7,8-dihydrofolate mutant F31S/Q35E, pH 7.6, 23°C Homo sapiens
1.5.1.3 10
-
 7,8-dihydrofolate wild-type, pH 7.6, 23°C Homo sapiens

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.5.1.3 0.000000031
-
 methotrexate wild-type, pH 7.6, 23°C Homo sapiens
1.5.1.3 0.0000017
-
 methotrexate mutant F31P, pH 7.6, 23°C Homo sapiens
1.5.1.3 0.000009
-
 methotrexate mutant F31G/Q35N, pH 7.6, 23°C Homo sapiens
1.5.1.3 0.00001
-
 methotrexate mutant F31P/Q35H, pH 7.6, 23°C Homo sapiens
1.5.1.3 0.000012
-
 methotrexate mutant F31P/Q35E, pH 7.6, 23°C Homo sapiens
1.5.1.3 0.000012
-
 methotrexate mutant F31R/F34A/Q35N, pH 7.6, 23°C Homo sapiens
1.5.1.3 0.000021
-
 methotrexate mutant F31R/Q35E, pH 7.6, 23°C Homo sapiens
1.5.1.3 0.00003
-
 methotrexate mutant F31S/Q35E, pH 7.6, 23°C Homo sapiens
1.5.1.3 0.000059
-
 methotrexate mutant F31R/F34T/Q35S, pH 7.6, 23°C Homo sapiens
1.5.1.3 0.000086
-
 methotrexate mutant F31R/F34T/Q35R, pH 7.6, 23°C Homo sapiens
1.5.1.3 0.00018
-
 methotrexate mutant F31A/F34V/Q35H, pH 7.6, 23°C Homo sapiens

IC50 Value

EC Number IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
1.5.1.3 0.000041
-
 wild-type, pH 7.6, 23°C Homo sapiens methotrexate
1.5.1.3 0.00011
-
 mutant F31P, pH 7.6, 23°C Homo sapiens methotrexate
1.5.1.3 0.00047
-
 mutant F31P/Q35E, pH 7.6, 23°C Homo sapiens methotrexate
1.5.1.3 0.00047
-
 mutant F31P/Q35H, pH 7.6, 23°C Homo sapiens methotrexate
1.5.1.3 0.00055
-
 mutant F31R/F34A/Q35N, pH 7.6, 23°C Homo sapiens methotrexate
1.5.1.3 0.00057
-
 mutant F31G/Q35N, pH 7.6, 23°C Homo sapiens methotrexate
1.5.1.3 0.00093
-
 mutant F31S/Q35E, pH 7.6, 23°C Homo sapiens methotrexate
1.5.1.3 0.0031
-
 mutant F31R/Q35E, pH 7.6, 23°C Homo sapiens methotrexate
1.5.1.3 0.0035
-
 mutant F31R/F34T/Q35R, pH 7.6, 23°C Homo sapiens methotrexate
1.5.1.3 0.0044
-
 mutant F31A/F34V/Q35H, pH 7.6, 23°C Homo sapiens methotrexate
1.5.1.3 0.0045
-
 mutant F31R/F34T/Q35S, pH 7.6, 23°C Homo sapiens methotrexate