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Literature summary for 6.3.5.4 extracted from

  • Duff, S.M.; Qi, Q.; Reich, T.; Wu, X.; Brown, T.; Crowley, J.H.; Fabbri, B.
    A kinetic comparison of asparagine synthetase isozymes from higher plants (2011), Plant Physiol. Biochem., 49, 251-256.
    View publication on PubMed
Search for more literature for 6.3.5.4

Application

Application Comment Organism
analysis development of a sensitive, non-radioactive assay for AsnS, based on incubation of desalted enzyme and substrates and then direct detection of either product asparagine or glutamate by HPLC Zea mays

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli Glycine max
expression in Escherichia coli Zea mays
gene asnS, expression of C-terminally His-tagged isozyme AsnS1 in Escherichia coli strains BL21(DE3) and Rosetta(DE3) mainly in the inclusion bodies Zea mays
gene asnS2, expression of nontagged isozyme ZmAsnS2 in Escherichia coli, expression of C-terminally His-tagged isozyme AsnS2 in Escherichia coli strains BL21(DE3) and Rosetta(DE3) mainly in the inclusion bodies although small amounts of ZmAsnS2 are recovered in the soluble fraction Zea mays
gene asnS3, expression of C-terminally His-tagged isozyme AsnS3 in Escherichia coli strains BL21(DE3) and Rosetta(DE3) mainly in the inclusion bodies Zea mays
gene asnS4, expression of C-terminally His-tagged isozyme AsnS4 in Escherichia coli strains BL21(DE3) and Rosetta(DE3) mainly in the inclusion bodies Zea mays

Inhibitors

Inhibitors Comment Organism Structure
asparagine
-
 Glycine max
asparagine competitive; competitive Zea mays
glutamate
-
 Glycine max
glutamate competitive; competitive Zea mays
L-asparagine competitive inhibition Zea mays
L-glutamate competitive inhibition Zea mays

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information the AsnS isozymes are kinetically distinct with substantial differences in Km (Gln) and Vmax values, overview. None of the enzymes has cooperative enzyme kinetics Zea mays
0.09
-
 L-glutamine pH 7.6, temperature not specified in the publication, recombinant His-tagged isozyme ZmAsn2 Zea mays
0.097
-
 ATP pH 7.6, 22°C Zea mays
0.097
-
 ATP pH 7.6, temperature not specified in the publication, recombinant His-tagged isozyme ZmAsn3 Zea mays
0.1 1 ATP pH 7.6, temperature not specified in the publication, recombinant His-tagged isozyme ZmAsn1 Zea mays
0.1 1 L-glutamine pH 7.6, temperature not specified in the publication, recombinant nontagged soluble isozyme ZmAsn2 Zea mays
0.1 2 L-aspartate pH 7.6, temperature not specified in the publication, recombinant His-tagged isozyme ZmAsn3 Zea mays
0.106
-
 ATP pH 7.6, 22°C Glycine max
0.11
-
 ATP pH 7.6, 22°C Zea mays
0.11
-
 L-Gln pH 7.6, 22°C Zea mays
0.125
-
 ATP pH 7.6, 22°C Zea mays
0.125
-
 ATP pH 7.6, temperature not specified in the publication, recombinant nontagged isozyme soluble ZmAsn2 Zea mays
0.128
-
 ATP pH 7.6, 22°C Zea mays
0.128
-
 ATP pH 7.6, temperature not specified in the publication, recombinant His-tagged isozyme ZmAsn2 Zea mays
0.128
-
 ATP pH 7.6, temperature not specified in the publication, recombinant His-tagged isozyme ZmAsn4 Zea mays
0.233
-
 L-Gln pH 7.6, 22°C Zea mays
0.233
-
 L-glutamine pH 7.6, temperature not specified in the publication, recombinant His-tagged isozyme ZmAsn4 Zea mays
0.254
-
 L-Gln pH 7.6, 22°C Glycine max
0.423
-
 L-Gln pH 7.6, 22°C Zea mays
0.423
-
 L-glutamine pH 7.6, temperature not specified in the publication, recombinant His-tagged isozyme ZmAsn3 Zea mays
0.543
-
 L-Gln pH 7.6, 22°C Zea mays
0.543
-
 L-glutamine pH 7.6, temperature not specified in the publication, recombinant His-tagged isozyme ZmAsn1 Zea mays
0.75
-
 NH3 pH 7.6, 22°C Zea mays
0.85
-
 L-Asp pH 7.6, 22°C Glycine max
0.9 1 L-aspartate pH 7.6, temperature not specified in the publication, recombinant nontagged soluble isozyme ZmAsn2 Zea mays
0.91
-
 L-Asp pH 7.6, 22°C Zea mays
0.93
-
 L-Asp pH 7.6, 22°C Zea mays
0.93
-
 L-aspartate pH 7.6, temperature not specified in the publication, recombinant His-tagged isozyme ZmAsn4 Zea mays
0.98
-
 L-Asp pH 7.6, 22°C Zea mays
0.98
-
 L-aspartate pH 7.6, temperature not specified in the publication, recombinant His-tagged isozyme ZmAsn1 Zea mays
0.98
-
 L-aspartate pH 7.6, temperature not specified in the publication, recombinant His-tagged isozyme ZmAsn2 Zea mays
1.2
-
 L-Asp pH 7.6, 22°C Zea mays
8.4
-
 NH3 pH 7.6, 22°C Zea mays
9
-
 NH3 pH 7.6, 22°C Zea mays
9.9
-
 NH3 pH 7.6, 22°C Zea mays

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Zea mays

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + L-aspartate + L-glutamine + H2O Zea mays
-
 AMP + diphosphate + L-asparagine + L-glutamate
-
 ?

Organism

Organism UniProt Comment Textmining
Glycine max
-
-
-
Zea mays B4FFJ0 isoform AsnS1
-
Zea mays B4FFJ0 isozyme AsnS1; isozyme AsnS1
-
Zea mays B5U8J7 isoform AsnS2
-
Zea mays B5U8J7 isozyme AsnS2; isozyme AsnS2
-
Zea mays B5U8J8 isoform AsnS3
-
Zea mays B5U8J8 isozyme AsnS3; isozyme AsnS3
-
Zea mays B5U8J9 isoform AsnS4
-
Zea mays B5U8J9 isozyme AsnS4; isozyme AsnS4
-

Purification (Commentary)

Purification (Comment) Organism
-
 Glycine max
-
 Zea mays
development of a method to refold recombinant AsnS from inclusion bodies Zea mays
refolded, solubilized recombinant His-tagged isozyme AsnS1 by nickel affinity chromatography Zea mays
refolded, solubilized recombinant His-tagged isozyme AsnS2 by nickel affinity chromatography, recombinant nontagged isozyme ZmAsnS2 from Escherichia coli by anion exchange chromatography Zea mays
refolded, solubilized recombinant His-tagged isozyme AsnS4 by nickel affinity chromatography Zea mays
refolded, solubilized recombinant His-tagged isozyme by nickel affinity chromatography Zea mays

Renatured (Commentary)

Renatured (Comment) Organism
refolding of recombinant C-terminally His-tagged enzyme from Escherichia coli strains BL21(DE3) and Rosetta(DE3) inclusion bodies to active proteins, method development using 6-8 M guanidine-HCl, overview. Vmax of the enzyme is lowered about 30% by refolding, but Km values for all three substrates show no substantial change Zea mays
refolding of recombinant C-terminally His-tagged enzymes from Escherichia coli strains BL21(DE3) and Rosetta(DE3) inclusion bodies to active proteins, method development using 6-8 M guanidine-HCl, overview. Vmax of the enzyme is lowered about 30% by refolding, but Km values for all three substrates show no substantial change Zea mays
refolding of recombinant C-terminally His-tagged isozyme AsnS1 from Escherichia coli strains BL21(DE3) and Rosetta(DE3) inclusion bodies to active protein, method development using 6-8 M guanidine-HCl, overview. Vmax of the enzyme is lowered about 30% by refolding, but Km values for all three substrates show no substantial change Zea mays

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + L-Asp + L-Gln
-
 Glycine max AMP + diphosphate + L-Asn + L-Glu
-
 ?
ATP + L-Asp + L-Gln
-
 Zea mays AMP + diphosphate + L-Asn + L-Glu
-
 ?
ATP + L-Asp + NH3
-
 Glycine max AMP + diphosphate + L-Asn
-
 ?
ATP + L-Asp + NH3
-
 Zea mays AMP + diphosphate + L-Asn
-
 ?
ATP + L-aspartate + L-glutamine + H2O
-
 Zea mays AMP + diphosphate + L-asparagine + L-glutamate
-
 ?

Synonyms

Synonyms Comment Organism
ASNS
-
 Zea mays
AsnS1
-
 Zea mays
AsnS2
-
 Zea mays
AsnS3
-
 Zea mays
AsnS4
-
 Zea mays
Asparagine synthetase
-
 Zea mays

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.6
-
 assay at Zea mays

Cofactor

Cofactor Comment Organism Structure
ATP
-
 Zea mays

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
0.25
-
 asparagine pH 7.6, 22°C Zea mays
0.25
-
 L-asparagine pH 7.6, temperature not specified in the publication, recombinant nontagged soluble isozyme ZmAsn2 Zea mays
0.3
-
 glutamate pH 7.6, 22°C Zea mays
0.3
-
 L-glutamate pH 7.6, temperature not specified in the publication, recombinant nontagged soluble isozyme ZmAsn2 Zea mays

IC50 Value

IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
1.1
-
 pH 7.6, 22°C Zea mays asparagine
1.1
-
 pH 7.6, temperature not specified in the publication, recombinant His-tagged isozyme ZmAsn1 Zea mays L-asparagine
1.2
-
 pH 7.6, 22°C Glycine max glutamate
1.2
-
 pH 7.6, 22°C Zea mays glutamate
1.2
-
 pH 7.6, temperature not specified in the publication, recombinant His-tagged isozyme ZmAsn4 Zea mays L-glutamate
1.3
-
 pH 7.6, 22°C Zea mays glutamate
1.3
-
 pH 7.6, 22°C Glycine max asparagine
1.3
-
 pH 7.6, 22°C Zea mays asparagine
1.3
-
 pH 7.6, temperature not specified in the publication, recombinant His-tagged isozyme ZmAs4 Zea mays L-asparagine
1.3
-
 pH 7.6, temperature not specified in the publication, recombinant His-tagged isozyme ZmAsn1 Zea mays L-glutamate
1.3
-
 pH 7.6, temperature not specified in the publication, recombinant His-tagged isozyme ZmAsn3 Zea mays L-glutamate
1.3
-
 pH 7.6, temperature not specified in the publication, recombinant nontagged soluble isozyme ZmAsn2 Zea mays L-glutamate
1.4
-
 pH 7.6, 22°C Zea mays asparagine
1.4
-
 pH 7.6, temperature not specified in the publication, recombinant nontagged soluble isozyme ZmAsn2 Zea mays L-asparagine
1.5
-
 pH 7.6, 22°C Zea mays asparagine
1.5
-
 pH 7.6, temperature not specified in the publication, recombinant His-tagged isozyme ZmAsn3 Zea mays L-asparagine

General Information

General Information Comment Organism
metabolism Asn is a major amino acid in maize and since AsnS is the primary means of Asn synthesis in plants it plays a very important role in nitrogen metabolism Zea mays