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Literature summary for 1.5.1.3 extracted from

  • Liu, C.T.; Francis, K.; Layfield, J.P.; Huang, X.; Hammes-Schiffer, S.; Kohen, A.; Benkovic, S.J.
    Escherichia coli dihydrofolate reductase catalyzed proton and hydride transfers temporal order and the roles of Asp27 and Tyr100 (2014), Proc. Natl. Acad. Sci. USA, 111, 18231-18236 .
    View publication on PubMedView publication on EuropePMC

Application

Application Comment Organism
drug development enzyme DHFR is an important drug target Escherichia coli

Protein Variants

Protein Variants Comment Organism
D27S site-directed mutagenesis, the mutant shows a 3400fold reduced rate for the NADPH-dependent reduction of 7,8-dihydrofolate at pH 7.0 in water compared to wild-type Escherichia coli
additional information competitive hydrogen to deuterium and hydrogen to tritium kinetic isotope effects (KIEs) on the second-order rate constant (kcat/Km) are measured for wild-type and mutant ecDHFR variants to assess the intrinsic KIE on the catalyzed hydride transfer Escherichia coli
Y100F site-directed mutagenesis, the mutant shows a 14fold reduced rate for the NADPH-dependent reduction of 7,8-dihydrofolate at pH 7.0 in water compared to wild-type Escherichia coli
Y100F/D27S site-directed mutagenesis, the mutant shows an over 100000fold reduced rate for the NADPH-dependent reduction of 7,8-dihydrofolate at pH 7.0 in water compared to wild-type Escherichia coli

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information kinetic behavior of the wild-type enzyme ecDHFR and enzyme mutants D27S, Y100F, and D27S/Y100F across an extended pH range, including pH 9.0. Competitive hydrogen to deuterium and hydrogen to tritium kinetic isotope effects (KIEs) on the second-order rate constant (kcat/Km) are measured for wild-type and mutant ecDHFR variants to assess the intrinsic KIE on the catalyzed hydride transfer. Presteady-state, Michaelis-Menten, and single-turnover kinetics Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
7,8-dihydrofolate + NADPH + H+ Escherichia coli
-
5,6,7,8-tetrahydrofolate + NADP+
-
r

Organism

Organism UniProt Comment Textmining
Escherichia coli P0ABQ4
-
-

Reaction

Reaction Comment Organism Reaction ID
5,6,7,8-tetrahydrofolate + NADP+ = 7,8-dihydrofolate + NADPH + H+ transfer of a proR hydride from the C4 atom of NADPH to the C6 position of the dihydropterin ring of DHF, mechanism of the proton and hydride transfer reaction, active site residues D27 and Y100 play a synergistic role in facilitating both the proton transfer and subsequent hydride transfer steps. Residue D27 appears to have a greater effect on the overall rate of conversion of DHF to tetrahydrofolate, Y100 plays an important electrostatic role in modulating the pKa of the N5 of DHF to enable the preprotonation of DHF by an active site water molecule. The D27 and Y100 residues function synergistically to provide an active site environment for the solvent-assisted protonation of N5 and to position the reacting substrates, NADPH and DHF, properly Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
7,8-dihydrofolate + NADPH + H+
-
Escherichia coli 5,6,7,8-tetrahydrofolate + NADP+
-
r

Synonyms

Synonyms Comment Organism
ecDHFR
-
Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Escherichia coli

pH Range

pH Minimum pH Maximum Comment Organism
additional information
-
analysis of the pH dependence of wild-type and mutant enzymes, overview. The differences between the temperature-independent deuteride transfer rate constant Dkhyd at pH 9.0 and temperature-dependent Dkhyd at pH 7.0 actually result from a pH-dependent commitment to catalysis on the khyd Escherichia coli
3.2 8.2 activity range Escherichia coli

Cofactor

Cofactor Comment Organism Structure
NADP+
-
Escherichia coli
NADPH
-
Escherichia coli

General Information

General Information Comment Organism
additional information analysis of the orientation of residues Y100, D27, M20, and the ligands in the active site of ecDHFR, PDB ID 1rx2 Escherichia coli