Application | Comment | Organism |
---|---|---|
drug development | enzyme DHFR is an important drug target | Escherichia coli |
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 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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P0ABQ4 | - |
- |
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 | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
7,8-dihydrofolate + NADPH + H+ | - |
Escherichia coli | 5,6,7,8-tetrahydrofolate + NADP+ | - |
r |
Synonyms | Comment | Organism |
---|---|---|
ecDHFR | - |
Escherichia coli |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Escherichia coli |
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 | Comment | Organism | Structure |
---|---|---|---|
NADP+ | - |
Escherichia coli | |
NADPH | - |
Escherichia coli |
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 |