BRENDA - Enzyme Database

Characterization of a bifunctional glyoxylate cycle enzyme, malate synthase/isocitrate lyase, of Euglena gracilis

Nakazawa, M.; Nishimura, M.; Inoue, K.; Ueda, M.; Inui, H.; Nakano, Y.; Miyatake, K.; J. Eukaryot. Microbiol. 58, 128-133 (2011)

Data extracted from this reference:

Activating Compound
EC Number
Activating Compound
Commentary
Organism
Structure
4.1.3.1
acetyl-CoA
increases the substrate binding affinities isocitrate and succinate, but had little effect on the affinity for glyoxylate
Euglena gracilis
Cloned(Commentary)
EC Number
Commentary
Organism
2.3.3.9
wild type enzyme is expressed in Escherichia coli BL21(DE3) cells, mutant enzymes are expressed in Escherichia coli ArcticExpress(DE3) RP cells
Euglena gracilis
4.1.3.1
His-tagged version (without signal peptide (amino acid residue 1-12)) expressed in Escherichia coli BL21(DE3)
Euglena gracilis
Engineering
EC Number
Amino acid exchange
Commentary
Organism
2.3.3.9
D475N
the glyoxylate cycle enzyme mutant completely loses malate synthase activity but retains isocitrate lyase activity
Euglena gracilis
2.3.3.9
additional information
the C-terminal domain of the glyoxylate cycle enzyme, when expressed alone (GCE(566-1165)), does not show any enzyme activity
Euglena gracilis
2.3.3.9
R187K
the glyoxylate cycle enzyme mutant completely loses malate synthase activity but retains isocitrate lyase activity
Euglena gracilis
4.1.3.1
D475N
putative acetyl-CoA binding site of malate synthase, no malate synthase activity, but isocitrate lyase activity
Euglena gracilis
4.1.3.1
additional information
truncated mutants: 13-573 (no isocitrate lyase activity, malate synthase activity), 566-1165 (no malate synthase activity), 20-573 (no isocitrate lyase activity, malate synthase activity), 38-573 (no isocitrate lyase activity), 20-1165 (isocitrate lyase activity and malate synthase activity), 38-1165
Euglena gracilis
4.1.3.1
R187K
putative acetyl-CoA binding site of malate synthase, no malate synthase activity, but isocitrate lyase activity
Euglena gracilis
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
2.3.3.9
0.065
-
glyoxylate
wild type full-length glyoxylate cycle enzyme, at 25°C, pH not specified in the publication
Euglena gracilis
2.3.3.9
0.073
-
glyoxylate
N-terminal malate synthase-active domain GCE(13-573) of glyoxylate cycle enzyme, at 25°C, pH not specified in the publication
Euglena gracilis
4.1.3.1
0.96
-
glyoxylate
D475N mutant protein, 0.15 mM acetyl-CoA, 10 mM succinate, pH 6.5, 30°C
Euglena gracilis
4.1.3.1
1.3
-
glyoxylate
D475N mutant protein, 10 mM succinate, pH 6.5, 30°C
Euglena gracilis
4.1.3.1
6
-
succinate
D475N mutant protein, 1.5 mM glyoxylate, sharply decreased with increasing acetyl-CoA concentration, pH 6.5, 30°C
Euglena gracilis
4.1.3.1
6.7
-
isocitrate
when acetyl-CoA is present at concentrations higher than 5 mM, the Km value for isocitrate decreased by about one-third and the Vmax value increased about 2-fold, pH not specified in the publication, temperature not specified in the publication
Euglena gracilis
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
2.3.3.9
62000
-
x * 62000, the 62000 Da N-terminal domain of glyoxylate cycle enzyme provides malate synthase activity, SDS-PAGE
Euglena gracilis
4.1.3.1
131000
-
4 * 131000, gel filtration, calculated subunit molecular mass
Euglena gracilis
4.1.3.1
530000
-
gel filtration
Euglena gracilis
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
2.3.3.9
Euglena gracilis
-
-
-
4.1.3.1
Euglena gracilis
Q8LPA6
-
-
Purification (Commentary)
EC Number
Commentary
Organism
2.3.3.9
DEAE Sepharose column chromatography, HisTrap HP column chromatography, and Superdex 200 pg gel filtration
Euglena gracilis
4.1.3.1
anion exchange chromatography (DEAE), immobilized metal ion affinity chromatography (Ni2+), gel filtration
Euglena gracilis
Source Tissue
EC Number
Source Tissue
Commentary
Organism
Textmining
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.3.3.9
glyoxylate + acetyl-CoA + H2O
-
715749
Euglena gracilis
(S)-malate + CoA
-
-
-
?
4.1.3.1
isocitrate
-
715749
Euglena gracilis
succinate + glyoxylate
-
-
-
r
Subunits
EC Number
Subunits
Commentary
Organism
2.3.3.9
?
x * 62000, the 62000 Da N-terminal domain of glyoxylate cycle enzyme provides malate synthase activity, SDS-PAGE
Euglena gracilis
4.1.3.1
homotetramer
4 * 131000, gel filtration, calculated subunit molecular mass
Euglena gracilis
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
2.3.3.9
14
-
glyoxylate
N-terminal malate synthase-active domain GCE(13-573) of glyoxylate cycle enzyme, at 25°C, pH not specified in the publication
Euglena gracilis
2.3.3.9
17
-
glyoxylate
wild type full-length glyoxylate cycle enzyme, at 25°C, pH not specified in the publication
Euglena gracilis
Activating Compound (protein specific)
EC Number
Activating Compound
Commentary
Organism
Structure
4.1.3.1
acetyl-CoA
increases the substrate binding affinities isocitrate and succinate, but had little effect on the affinity for glyoxylate
Euglena gracilis
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
2.3.3.9
wild type enzyme is expressed in Escherichia coli BL21(DE3) cells, mutant enzymes are expressed in Escherichia coli ArcticExpress(DE3) RP cells
Euglena gracilis
4.1.3.1
His-tagged version (without signal peptide (amino acid residue 1-12)) expressed in Escherichia coli BL21(DE3)
Euglena gracilis
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
2.3.3.9
D475N
the glyoxylate cycle enzyme mutant completely loses malate synthase activity but retains isocitrate lyase activity
Euglena gracilis
2.3.3.9
additional information
the C-terminal domain of the glyoxylate cycle enzyme, when expressed alone (GCE(566-1165)), does not show any enzyme activity
Euglena gracilis
2.3.3.9
R187K
the glyoxylate cycle enzyme mutant completely loses malate synthase activity but retains isocitrate lyase activity
Euglena gracilis
4.1.3.1
D475N
putative acetyl-CoA binding site of malate synthase, no malate synthase activity, but isocitrate lyase activity
Euglena gracilis
4.1.3.1
additional information
truncated mutants: 13-573 (no isocitrate lyase activity, malate synthase activity), 566-1165 (no malate synthase activity), 20-573 (no isocitrate lyase activity, malate synthase activity), 38-573 (no isocitrate lyase activity), 20-1165 (isocitrate lyase activity and malate synthase activity), 38-1165
Euglena gracilis
4.1.3.1
R187K
putative acetyl-CoA binding site of malate synthase, no malate synthase activity, but isocitrate lyase activity
Euglena gracilis
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
2.3.3.9
0.065
-
glyoxylate
wild type full-length glyoxylate cycle enzyme, at 25°C, pH not specified in the publication
Euglena gracilis
2.3.3.9
0.073
-
glyoxylate
N-terminal malate synthase-active domain GCE(13-573) of glyoxylate cycle enzyme, at 25°C, pH not specified in the publication
Euglena gracilis
4.1.3.1
0.96
-
glyoxylate
D475N mutant protein, 0.15 mM acetyl-CoA, 10 mM succinate, pH 6.5, 30°C
Euglena gracilis
4.1.3.1
1.3
-
glyoxylate
D475N mutant protein, 10 mM succinate, pH 6.5, 30°C
Euglena gracilis
4.1.3.1
6
-
succinate
D475N mutant protein, 1.5 mM glyoxylate, sharply decreased with increasing acetyl-CoA concentration, pH 6.5, 30°C
Euglena gracilis
4.1.3.1
6.7
-
isocitrate
when acetyl-CoA is present at concentrations higher than 5 mM, the Km value for isocitrate decreased by about one-third and the Vmax value increased about 2-fold, pH not specified in the publication, temperature not specified in the publication
Euglena gracilis
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
2.3.3.9
62000
-
x * 62000, the 62000 Da N-terminal domain of glyoxylate cycle enzyme provides malate synthase activity, SDS-PAGE
Euglena gracilis
4.1.3.1
131000
-
4 * 131000, gel filtration, calculated subunit molecular mass
Euglena gracilis
4.1.3.1
530000
-
gel filtration
Euglena gracilis
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
2.3.3.9
DEAE Sepharose column chromatography, HisTrap HP column chromatography, and Superdex 200 pg gel filtration
Euglena gracilis
4.1.3.1
anion exchange chromatography (DEAE), immobilized metal ion affinity chromatography (Ni2+), gel filtration
Euglena gracilis
Source Tissue (protein specific)
EC Number
Source Tissue
Commentary
Organism
Textmining
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.3.3.9
glyoxylate + acetyl-CoA + H2O
-
715749
Euglena gracilis
(S)-malate + CoA
-
-
-
?
4.1.3.1
isocitrate
-
715749
Euglena gracilis
succinate + glyoxylate
-
-
-
r
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
2.3.3.9
?
x * 62000, the 62000 Da N-terminal domain of glyoxylate cycle enzyme provides malate synthase activity, SDS-PAGE
Euglena gracilis
4.1.3.1
homotetramer
4 * 131000, gel filtration, calculated subunit molecular mass
Euglena gracilis
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
2.3.3.9
14
-
glyoxylate
N-terminal malate synthase-active domain GCE(13-573) of glyoxylate cycle enzyme, at 25°C, pH not specified in the publication
Euglena gracilis
2.3.3.9
17
-
glyoxylate
wild type full-length glyoxylate cycle enzyme, at 25°C, pH not specified in the publication
Euglena gracilis