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Literature summary for 2.7.2.4 extracted from

  • Curien, G.; Laurencin, M.; Robert-Genthon, M.; Dumas, R.
    Allosteric monofunctional aspartate kinases from Arabidopsis (2007), FEBS J., 274, 164-176.
    View publication on PubMed

Application

Application Comment Organism
additional information upon binding to the inactive AK1-Lys complex, S-adenosyl-L-methionine promotes a slow conformational transition leading to formation of a stable aspartate kinase 1-Lys-S-adenosyl-L-methionine complex. Increase in AK1 apparent affinity for lysine in the presence of S-adenosyl-L-methionine results from the displacement of the unfavorable equilibrium between AK1 and aspartate kinase 1-Lys towards the inactive form, S-adenosyl-L-methionine and Lys binding to AK1 is sequential, with Lys binding preceding S-adenosyl-L-methionine binding Arabidopsis thaliana
additional information upon binding to the inactive AK1–Lys complex, S-adenosyl-l-methionine promotes a slow conformational transition leading to formation of a stable aspartate kinase 1–Lys–S-adenosyl-l-methionine complex. Increase in AK1 apparent affinity for lysine in the presence of S-adenosyl-l-methionine results from the displacement of the unfavorable equilibrium between AK1 and aspartate kinase 1–Lys towards the inactive form, S-adenosyl-l-methionine and Lys binding to AK1 is sequential, with Lys binding preceding S-adenosyl-l-methionine binding Arabidopsis thaliana

Cloned(Commentary)

Cloned (Comment) Organism
ligated into pET 23d(+) vector and Escherichia coli strain DH10B for cloning, expression in Escherichia coli BL21 (DE3) pLysS codon+ Arabidopsis thaliana
ligated into pET 23d(+) vector, expressed in Escherichia coli BL21 codon (+) Arabidopsis thaliana

Inhibitors

Inhibitors Comment Organism Structure
L-lysine AK1 is inhibited in a synergistic manner by lysine and S-adenosyl-L-methionine, in the absence of S-adenosyl-L-methionine AK1 displays low apparent affinity for lysine compared to AK2 and AK3; AK1 is inhibited in a synergistic manner by lysine and SAM; AK2 is inhibited by lysine, SAM by itself has no effect on the enzyme activity; AK3 is inhibited only by lysine, SAM by itself has no effect on the enzyme activity Arabidopsis thaliana
additional information no additional inhibition is observed upon addition of Thr or Leu in the presence of 100 microM Lys and 20 microM SAM; no additional inhibition is observed upon addition of Thr or Leu in the presence of 5 microM Lys for AK2; no additional inhibition is observed upon addition of Thr or Leu in the presence of 5 microM Lys for AK3; the other amino acids tested (Met, Gln, Asn, Glu, Arg) have no effect on the enzyme activities at 2.5 mM either in the presence or the absence of the inhibitor Lys or Lys plus SAM Arabidopsis thaliana
S-adenosyl-L-methionine AK1 is inhibited in a synergistic manner by lysine and S-adenosyl-L-methionine, in the presence of S-adenosyl-L-methionine, the apparent affinity of AK1 for lysine increases considerably for lysine inhibition similar to those of AK2 and AK3 Arabidopsis thaliana

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.56
-
ATP
-
Arabidopsis thaliana
0.56
-
ATP AK3, 30°C, 50 mM HEPES, pH 8.0, 150 mM KCl, 20 mM MgCl2 with a coupled assay using aspartate semialdehyde dehydrogenase from Arabidopsis thaliana Arabidopsis thaliana
0.98
-
ATP AK2, 30°C, 50 mM HEPES, pH 8.0, 150 mM KCl, 20 mM MgCl2 with a coupled assay using aspartate semialdehyde dehydrogenase from Arabidopsis thaliana Arabidopsis thaliana
1.095
-
L-aspartate
-
Arabidopsis thaliana
1.095
-
aspartate AK3, 30°C, 50 mM HEPES, pH 8.0, 150 mM KCl, 20 mM MgCl2 with a coupled assay using aspartate semialdehyde dehydrogenase from Arabidopsis thaliana Arabidopsis thaliana
1.7
-
ATP
-
Arabidopsis thaliana
1.7
-
ATP 30°C, 50 mM HEPES, pH 8.0, 150 mM KCl, 20 mM MgCl2 with a coupled assay using aspartate semialdehyde dehydrogenase from Arabidopsis thaliana Arabidopsis thaliana
1.94
-
aspartate AK2, 30°C, 50 mM HEPES, pH 8.0, 150 mM KCl, 20 mM MgCl2 with a coupled assay using aspartate semialdehyde dehydrogenase from Arabidopsis thaliana Arabidopsis thaliana
2.037
-
L-aspartate
-
Arabidopsis thaliana
2.037
-
aspartate 30°C, 50 mM HEPES, pH 8.0, 150 mM KCl, 20 mM MgCl2 with a coupled assay using aspartate semialdehyde dehydrogenase from Arabidopsis thaliana Arabidopsis thaliana

Localization

Localization Comment Organism GeneOntology No. Textmining
chloroplast
-
Arabidopsis thaliana 9507
-

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
53200
-
gel filtration Arabidopsis thaliana
53200
-
monofunctional AK2, proteins are separated on a 10% polyacrylamide (w/v) slab gel under denaturing conditions and stained with Coomassie brilliant blue R-250 Arabidopsis thaliana
55100
-
gel filtration Arabidopsis thaliana
55100
-
monofunctional AK3, proteins are separated on a 10% polyacrylamide (w/v) slab gel under denaturing conditions and stained with Coomassie brilliant blue R-250 Arabidopsis thaliana
55900
-
gel filtration Arabidopsis thaliana
55900
-
monofunctional AK1, proteins are separated on a 10% polyacrylamide (w/v) slab gel under denaturing conditions and stained with Coomassie brilliant blue R-250 Arabidopsis thaliana

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + L-aspartate Arabidopsis thaliana
-
ADP + 4-phospho-L-aspartate
-
?

Organism

Organism UniProt Comment Textmining
Arabidopsis thaliana
-
-
-
Arabidopsis thaliana O23653 ligated into pET 23d(+) vector and Escherichia coli strain DH10B for cloning, expression in Escherichia coli BL21 (DE3) pLysS codon+
-
Arabidopsis thaliana Q9LYU8
-
-
Arabidopsis thaliana Q9LYU8 ligated into pET 23d(+) vector and Escherichia coli strain DH10B for cloning, expression in Escherichia coli BL21 (DE3) pLysS codon+
-
Arabidopsis thaliana Q9S702
-
-
Arabidopsis thaliana Q9S702 ligated into pET 23d(+) vector and Escherichia coli strain DH10B for cloning, expression in Escherichia coli BL21 (DE3) pLysS codon+
-

Purification (Commentary)

Purification (Comment) Organism
on anion exchange column and by gel filtration Arabidopsis thaliana
weak anion exchange chromatography, affinity chromatogrphy for AK1 Arabidopsis thaliana
weak anion exchange chromatography, gel filtration Arabidopsis thaliana

Storage Stability

Storage Stability Organism
-80°C, HEPES buffer, pH 7.5, 1 mM Lys, 10% (v/v) glycerol, several month, without any loss of activity Arabidopsis thaliana
-80°C, HEPES buffer, pH 7.5, 1 mm Lys, 10% (v/v) glycerol, several months Arabidopsis thaliana

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + L-aspartate
-
Arabidopsis thaliana ADP + 4-phospho-L-aspartate
-
?

Synonyms

Synonyms Comment Organism
AK1
-
Arabidopsis thaliana
AK2
-
Arabidopsis thaliana
AK3
-
Arabidopsis thaliana
aspartate kinase
-
Arabidopsis thaliana
aspartate kinase 1
-
Arabidopsis thaliana
aspartate kinase 2
-
Arabidopsis thaliana
aspartate kinase 3
-
Arabidopsis thaliana

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
additional information
-
AK3 is highly unstable when stored at room temperature, losing 95% of its activity in 24 h Arabidopsis thaliana
additional information
-
AK1 retaines all activity when stored at room temperature Arabidopsis thaliana
additional information
-
AK2 retaines all activity when stored at room temperature Arabidopsis thaliana
additional information
-
AK3 proves to be highly unstable when stored at room temperature, losing 95% of its activity in 24 h Arabidopsis thaliana
20
-
AK3 is highly unstable when stored at room temperature, losing 95% of its activity in 24 h. During this period of time, AK1 and AK2 retained all their activity Arabidopsis thaliana
20
-
in 24 h, AK1 retains all its activity Arabidopsis thaliana

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
8.4
-
ATP
-
Arabidopsis thaliana
8.4
-
ATP monofunctional AK3 Arabidopsis thaliana
14.5
-
ATP
-
Arabidopsis thaliana
14.5
-
ATP monofunctional AK2 Arabidopsis thaliana
23.4
-
ATP
-
Arabidopsis thaliana
23.4
-
ATP monofunctional AK1 Arabidopsis thaliana