Activating Compound | Comment | Organism | Structure |
---|---|---|---|
additional information | cannot be activated by fumarate | Homo sapiens | |
additional information | no activation by fumarate | Homo sapiens |
Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli BL21(DE3) cells | Homo sapiens |
recombinant C-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, dialysis, and ultrafiltration | Homo sapiens |
Crystallization (Comment) | Organism |
---|---|
hanging drop vapor diffusion method, using 250 mM LiCl and 19% (w/v) PEG 3350 | Homo sapiens |
purified apoenzyme, hanging drop vapor diffusion method, 6 mg/ml protein is crystallized against a reservoir solution containing 250 mM LiCl and 19% w/v PEG 3350, room temperature, 5-7 days, X-ray diffraction structure determination and analysis at 2.55 A resolution, molecular replacement using the structure of pigeon c-NADP-ME in complex with NADP+, Mn2+, and oxalate as search model, modelling | Homo sapiens |
Protein Variants | Comment | Organism |
---|---|---|
E73K | site-directed mutagenesis | Homo sapiens |
E73K | the mutant shows decreased turnover numbers for (S)-malate and NADP+ compared to the wild type enzyme | Homo sapiens |
K57S/E59N/K73E/D102S | site-directed mutagenesis, the mutant is primarily monomeric with some dimer formation | Homo sapiens |
additional information | multiple residues corresponding to the fumarate-binding site are mutated in human c-NADP-ME to correspond to those found in human m-NAD(P)-ME, EC 1.1.1.39. No significant difference between the wild-type and mutant enzymes in Km values for NADP+ and malate, and in kcat values. A chimeric enzyme, [51-105]_c-NADP-ME, is designed to include the putative fumarate-binding site ofm-NAD(P)-ME at the dimer interface of c-NADP-ME, but the chimera remains nonallosteric | Homo sapiens |
N59E | site-directed mutagenesis | Homo sapiens |
N59E | the mutant shows decreased turnover numbers for (S)-malate and NADP+ compared to the wild type enzyme | Homo sapiens |
N59E/E73K | site-directed mutagenesis | Homo sapiens |
N59E/E73K | the mutant shows decreased turnover numbers for (S)-malate and NADP+ compared to the wild type enzyme | Homo sapiens |
N59E/E73K/S102D | site-directed mutagenesis | Homo sapiens |
N59E/E73K/S102D | the mutant shows decreased turnover numbers for (S)-malate and NADP+ compared to the wild type enzyme | Homo sapiens |
S102D | site-directed mutagenesis | Homo sapiens |
S102D | the mutant shows decreased turnover numbers for (S)-malate and NADP+ compared to the wild type enzyme | Homo sapiens |
S57K | site-directed mutagenesis | Homo sapiens |
S57K | the mutant shows increased turnover numbers for (S)-malate and NADP+ compared to the wild type enzyme | Homo sapiens |
S57K/N59E/E73K | site-directed mutagenesis | Homo sapiens |
S57K/N59E/E73K | the mutant shows wild type turnover numbers for (S)-malate and NADP+ | Homo sapiens |
S57K/N59E/E73K/S102D | the mutant shows increased turnover numbers for (S)-malate and NADP+ compared to the wild type enzyme | Homo sapiens |
S57K/N59E/E73K/S102D | site-directed mutagenesis, the mutant is tetrameric | Homo sapiens |
S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G | the mutant shows decreased turnover numbers for (S)-malate and NADP+ compared to the wild type enzyme | Homo sapiens |
S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G | site-directed mutagenesis, the mutant is tetrameric | Homo sapiens |
S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G/D90E/K106S/Q121S/L125H | site-directed mutagenesis | Homo sapiens |
S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G/D90E/K106S/Q121S/L125H | the mutant shows decreased turnover numbers for (S)-malate and NADP+ compared to the wild type enzyme | Homo sapiens |
S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G/K106S/Q121S/L125H | site-directed mutagenesis | Homo sapiens |
S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G/K106S/Q121S/L125H | the mutant shows decreased turnover numbers for (S)-malate and NADP+ compared to the wild type enzyme | Homo sapiens |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0018 | - |
NADP+ | mutant enzyme S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G/D90E/K106S/Q121S/L125H, at pH 7.4 and 30°C | Homo sapiens | |
0.0018 | - |
NADPH | pH 7.4, 30°C, recombinant mutant S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G/D90E/K106S/Q121S/L125H | Homo sapiens | |
0.0019 | - |
NADP+ | wild type enzyme, at pH 7.4 and 30°C | Homo sapiens | |
0.0019 | - |
NADP+ | mutant enzyme S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G, at pH 7.4 and 30°C | Homo sapiens | |
0.0019 | - |
NADPH | pH 7.4, 30°C, recombinant mutant 57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G | Homo sapiens | |
0.0019 | - |
NADPH | pH 7.4, 30°C, recombinant wild-type enzyme | Homo sapiens | |
0.0023 | - |
NADP+ | mutant enzyme S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G/K106S/Q121S/L125H, at pH 7.4 and 30°C | Homo sapiens | |
0.0023 | - |
NADPH | pH 7.4, 30°C, recombinant mutant S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G/K106S/Q121S/L125H | Homo sapiens | |
0.0027 | - |
NADPH | pH 7.4, 30°C, recombinant mutant [51-105]_c-NADP-ME | Homo sapiens | |
0.0031 | - |
NADP+ | mutant enzyme S57K/N59E/E73K/S102D, at pH 7.4 and 30°C | Homo sapiens | |
0.0031 | - |
NADPH | pH 7.4, 30°C, recombinant mutant S57K/N59E/E73K/S102D | Homo sapiens | |
0.0045 | - |
NADP+ | mutant enzyme N59E/E73K/S102D, at pH 7.4 and 30°C | Homo sapiens | |
0.0045 | - |
NADP+ | mutant enzyme S57K, at pH 7.4 and 30°C | Homo sapiens | |
0.0045 | - |
NADPH | pH 7.4, 30°C, recombinant mutant N59E/E73K/S102D | Homo sapiens | |
0.0045 | - |
NADPH | pH 7.4, 30°C, recombinant mutant S57K | Homo sapiens | |
0.0046 | - |
NADP+ | mutant enzyme N59E/E73K, at pH 7.4 and 30°C | Homo sapiens | |
0.0046 | - |
NADPH | pH 7.4, 30°C, recombinant mutant N59E/E73K | Homo sapiens | |
0.0049 | - |
NADP+ | mutant enzyme S102D, at pH 7.4 and 30°C | Homo sapiens | |
0.0049 | - |
NADPH | pH 7.4, 30°C, recombinant mutant S102D | Homo sapiens | |
0.0069 | - |
NADP+ | mutant enzyme S57K/N59E/E73K, at pH 7.4 and 30°C | Homo sapiens | |
0.0069 | - |
NADPH | pH 7.4, 30°C, recombinant mutant S57K/N59E/E73K | Homo sapiens | |
0.0089 | - |
NADP+ | mutant enzyme N59E, at pH 7.4 and 30°C | Homo sapiens | |
0.0089 | - |
NADPH | pH 7.4, 30°C, recombinant mutant N59E | Homo sapiens | |
0.0125 | - |
NADP+ | mutant enzyme E73K, at pH 7.4 and 30°C | Homo sapiens | |
0.0125 | - |
NADPH | pH 7.4, 30°C, recombinant mutant E73K | Homo sapiens | |
0.8 | - |
(S)-malate | mutant enzyme S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G, at pH 7.4 and 30°C | Homo sapiens | |
0.8 | - |
(S)-malate | mutant enzyme S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G/K106S/Q121S/L125H, at pH 7.4 and 30°C | Homo sapiens | |
0.8 | - |
(S)-malate | pH 7.4, 30°C, recombinant mutant 57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G | Homo sapiens | |
0.8 | - |
(S)-malate | pH 7.4, 30°C, recombinant mutant S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G/K106S/Q121S/L125H | Homo sapiens | |
0.8 | - |
(S)-malate | pH 7.4, 30°C, recombinant mutant [51-105]_c-NADP-ME | Homo sapiens | |
0.9 | - |
(S)-malate | mutant enzyme N59E, at pH 7.4 and 30°C | Homo sapiens | |
0.9 | - |
(S)-malate | pH 7.4, 30°C, recombinant mutant N59E | Homo sapiens | |
1 | - |
(S)-malate | mutant enzyme S57K/N59E/E73K, at pH 7.4 and 30°C | Homo sapiens | |
1 | - |
(S)-malate | mutant enzyme S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G/D90E/K106S/Q121S/L125H, at pH 7.4 and 30°C | Homo sapiens | |
1 | - |
(S)-malate | pH 7.4, 30°C, recombinant mutant S57K/N59E/E73K | Homo sapiens | |
1 | - |
(S)-malate | pH 7.4, 30°C, recombinant mutant S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G/D90E/K106S/Q121S/L125H | Homo sapiens | |
1.2 | - |
(S)-malate | wild type enzyme, at pH 7.4 and 30°C | Homo sapiens | |
1.2 | - |
(S)-malate | mutant enzyme N59E/E73K, at pH 7.4 and 30°C | Homo sapiens | |
1.2 | - |
(S)-malate | mutant enzyme S57K/N59E/E73K/S102D, at pH 7.4 and 30°C | Homo sapiens | |
1.2 | - |
(S)-malate | pH 7.4, 30°C, recombinant mutant N59E/E73K | Homo sapiens | |
1.2 | - |
(S)-malate | pH 7.4, 30°C, recombinant wild-type enzyme | Homo sapiens | |
1.3 | - |
(S)-malate | mutant enzyme E73K, at pH 7.4 and 30°C | Homo sapiens | |
1.3 | - |
(S)-malate | pH 7.4, 30°C, recombinant mutant E73K | Homo sapiens | |
1.3 | - |
(S)-malate | pH 7.4, 30°C, recombinant mutant S57K/N59E/E73K/S102D | Homo sapiens | |
1.4 | - |
(S)-malate | mutant enzyme N59E/E73K/S102D, at pH 7.4 and 30°C | Homo sapiens | |
1.4 | - |
(S)-malate | pH 7.4, 30°C, recombinant mutant N59E/E73K/S102D | Homo sapiens | |
1.6 | - |
(S)-malate | mutant enzyme S102D, at pH 7.4 and 30°C | Homo sapiens | |
1.6 | - |
(S)-malate | pH 7.4, 30°C, recombinant mutant S102D | Homo sapiens | |
1.7 | - |
(S)-malate | mutant enzyme S57K, at pH 7.4 and 30°C | Homo sapiens | |
1.7 | - |
(S)-malate | pH 7.4, 30°C, recombinant mutant S57K | Homo sapiens |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
cytosol | - |
Homo sapiens | 5829 | - |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | activates | Homo sapiens | |
Mg2+ | dependent on | Homo sapiens |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
(S)-malate + NADP+ | Homo sapiens | - |
pyruvate + CO2 + NADPH | - |
r | |
(S)-malate + NADP+ | Homo sapiens | - |
pyruvate + CO2 + NADPH + H+ | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Homo sapiens | P48163 | - |
- |
Purification (Comment) | Organism |
---|---|
Ni-NTA column chromatography | Homo sapiens |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(S)-malate + NADP+ | - |
Homo sapiens | pyruvate + CO2 + NADPH | - |
r | |
(S)-malate + NADP+ | - |
Homo sapiens | pyruvate + CO2 + NADPH + H+ | - |
? |
Subunits | Comment | Organism |
---|---|---|
homotetramer | x-ray crystallography | Homo sapiens |
More | structure comparisons, overview | Homo sapiens |
tetramer | a dimer of dimers. The c-NADP-ME monomer is composed of four domains. Domain A (residues 23-130) is predominantly helical and confers the ability to bind fumarate. Domain B (residues 131-277 and 464-535) contains a central five-stranded parallel beta-sheet surrounded by helices on both sides. Domain C (residues 278-463) exhibits a dinucleotide-binding Rossmann fold with a modification: strand beta3 is replaced by a short antiparallel beta-strand. Domain D (residues 536-579) contains one helix followed by a long extended random coil structure protruding away from the ordered portion of the ME monomer. The active site of ME is located at the interface of domains B and C, whereas residues in domains A and D primarily participate in the formation of dimers and tetramers | Homo sapiens |
Synonyms | Comment | Organism |
---|---|---|
c-NADP-ME | - |
Homo sapiens |
cytosolic NADP+-dependent malic enzyme | - |
Homo sapiens |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Homo sapiens |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
2 | 8 | (S)-malate | mutant enzyme N59E/E73K, at pH 7.4 and 30°C | Homo sapiens | |
2 | 8 | (S)-malate | mutant enzyme N59E/E73K/S102D, at pH 7.4 and 30°C | Homo sapiens | |
2 | 8 | (S)-malate | pH 7.4, 30°C, recombinant mutant N59E/E73K | Homo sapiens | |
2 | 8 | (S)-malate | pH 7.4, 30°C, recombinant mutant N59E/E73K/S102D | Homo sapiens | |
3 | 6 | (S)-malate | pH 7.4, 30°C, recombinant mutant [51-105]_c-NADP-ME | Homo sapiens | |
21 | - |
(S)-malate | mutant enzyme S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G/D90E/K106S/Q121S/L125H, at pH 7.4 and 30°C | Homo sapiens | |
21 | - |
(S)-malate | pH 7.4, 30°C, recombinant mutant S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G/D90E/K106S/Q121S/L125H | Homo sapiens | |
22 | - |
(S)-malate | mutant enzyme S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G, at pH 7.4 and 30°C | Homo sapiens | |
22 | - |
(S)-malate | pH 7.4, 30°C, recombinant mutant 57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G | Homo sapiens | |
23 | - |
(S)-malate | mutant enzyme S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G/K106S/Q121S/L125H, at pH 7.4 and 30°C | Homo sapiens | |
23 | - |
(S)-malate | pH 7.4, 30°C, recombinant mutant S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G/K106S/Q121S/L125H | Homo sapiens | |
27 | - |
(S)-malate | mutant enzyme N59E, at pH 7.4 and 30°C | Homo sapiens | |
27 | - |
(S)-malate | pH 7.4, 30°C, recombinant mutant N59E | Homo sapiens | |
29 | - |
(S)-malate | mutant enzyme S102D, at pH 7.4 and 30°C | Homo sapiens | |
29 | - |
(S)-malate | pH 7.4, 30°C, recombinant mutant S102D | Homo sapiens | |
30 | - |
(S)-malate | wild type enzyme, at pH 7.4 and 30°C | Homo sapiens | |
30 | - |
(S)-malate | mutant enzyme E73K, at pH 7.4 and 30°C | Homo sapiens | |
30 | - |
(S)-malate | mutant enzyme S57K/N59E/E73K, at pH 7.4 and 30°C | Homo sapiens | |
30 | - |
(S)-malate | pH 7.4, 30°C, recombinant mutant E73K | Homo sapiens | |
30 | - |
(S)-malate | pH 7.4, 30°C, recombinant mutant S57K/N59E/E73K | Homo sapiens | |
30 | - |
(S)-malate | pH 7.4, 30°C, recombinant wild-type enzyme | Homo sapiens | |
31 | - |
(S)-malate | mutant enzyme S57K/N59E/E73K/S102D, at pH 7.4 and 30°C | Homo sapiens | |
31 | - |
(S)-malate | pH 7.4, 30°C, recombinant mutant S57K/N59E/E73K/S102D | Homo sapiens | |
32 | - |
(S)-malate | mutant enzyme S57K, at pH 7.4 and 30°C | Homo sapiens | |
32 | - |
(S)-malate | pH 7.4, 30°C, recombinant mutant S57K | Homo sapiens |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.4 | - |
assay at | Homo sapiens |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NADP+ | - |
Homo sapiens | |
NADPH | - |
Homo sapiens |
General Information | Comment | Organism |
---|---|---|
additional information | the molecular basis for the different allosteric properties and quaternary structural stability of m-NAD(P)-ME, EC 1.1.1.39 and c-NADP-ME, EC 1.1.1.40. The structural features near the fumarate binding site and the dimer interface are highly related to the quaternary structural stability of c-NADP-ME and m-NAD(P)-ME | Homo sapiens |