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Literature summary extracted from

  • Dang, T.; Ingram-Smith, C.
    Investigation of pyrophosphate versus ATP substrate selection in the Entamoeba histolytica acetate kinase (2017), Sci. Rep., 7, 5912 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.7.2.1 expressed in Escherichia coli Rosetta2 (DE3) pLysS cells Methanosarcina thermophila
2.7.2.12 expressed in Escherichia coli YBS121 DELTAack DELTApta cells Entamoeba histolytica

Protein Variants

EC Number Protein Variants Comment Organism
2.7.2.1 G331Q/I332M the mutations result in substantial reductions in kcat compared to the wild type enzyme. In the acetate-forming direction, catalysis is reduced over 100fold, and in the acetyl phosphate-forming direction, kcat is reduced about 50fold. This alteration results in about 5fold increase in Km for ADP and ATP, and a 15fold increase in Km for acetate but no substantial change in the Km for acetyl phosphate Methanosarcina thermophila
2.7.2.12 D272A/R274A/Q323G/M324I the mutations decrease kcat in the acetate-forming direction by about 2500-5000fold but have little effect on Km for either substrate compared to the wild type enzyme. This mutant has no detectable activity in the acetyl phosphate-forming direction Entamoeba histolytica
2.7.2.12 DELTAG203/Q323G/M324I inactive Entamoeba histolytica
2.7.2.12 Q323A/M324A the variant displays similar Km values for acetyl phosphate and slightly decreased Km values for phosphate as the wild type enzyme but the kcat value is decreased 19fold resulting in about 11fold reduced catalytic efficiency. In the direction of acetyl phosphate formation, these variant displays slightly increased Km for acetate but no increase in Km for diphosphate and only mild decrease in kcat Entamoeba histolytica
2.7.2.12 Q323G/M324I the variant displays similar Km values for acetyl phosphate and slightly decreased Km values for phosphate as the wild type enzyme but the kcat value is decreased 8.3fold resulting in about 7fold reduced catalytic efficiency. In the direction of acetyl phosphate formation, these variant displays slightly increased Km for acetate but no increase in Km for diphosphate and only mild decrease in kcat Entamoeba histolytica

Inhibitors

EC Number Inhibitors Comment Organism Structure
2.7.2.1 diphosphate about 70% inhibition in the acetate-forming direction and about 90% inhibition in the acetyl phosphate-forming direction Methanosarcina thermophila
2.7.2.1 additional information not inhibited by phosphate Methanosarcina thermophila
2.7.2.12 ADP 80% inhibition at 1 mM Entamoeba histolytica
2.7.2.12 ATP about 30% inhibition at 1 mM Entamoeba histolytica
2.7.2.12 additional information not inhibited by AMP Entamoeba histolytica

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.7.2.1 1.2
-
 acetyl phosphate wild type enzyme, at pH 7.0 and 37°C Methanosarcina thermophila
2.7.2.1 1.7
-
 acetyl phosphate mutant enzyme G331Q/I332M, at pH 7.0 and 37°C Methanosarcina thermophila
2.7.2.1 1.7
-
 ATP wild type enzyme, at pH 5.5 and 45°C Methanosarcina thermophila
2.7.2.1 1.8
-
 ADP wild type enzyme, at pH 7.0 and 37°C Methanosarcina thermophila
2.7.2.1 5.7
-
 ADP mutant enzyme G331Q/I332M, at pH 7.0 and 37°C Methanosarcina thermophila
2.7.2.1 9.4
-
 ATP mutant enzyme G331Q/I332M, at pH 5.5 and 45°C Methanosarcina thermophila
2.7.2.1 20
-
 acetate wild type enzyme, at pH 5.5 and 45°C Methanosarcina thermophila
2.7.2.1 302
-
 acetate mutant enzyme G331Q/I332M, at pH 5.5 and 45°C Methanosarcina thermophila
2.7.2.12 0.34
-
 acetyl phosphate mutant enzyme Q323A/M324A, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 0.47
-
 acetyl phosphate mutant enzyme Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 0.57
-
 acetyl phosphate wild type enzyme, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 0.66
-
 acetyl phosphate mutant enzyme D272A/R274A/Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 2
-
 diphosphate mutant enzyme Q323G/M324I, at pH 5.5 and 45°C Entamoeba histolytica
2.7.2.12 2.1
-
 diphosphate wild type enzyme, at pH 5.5 and 45°C Entamoeba histolytica
2.7.2.12 2.3
-
 diphosphate mutant enzyme Q323A/M324A, at pH 5.5 and 45°C Entamoeba histolytica
2.7.2.12 7.3
-
 phosphate mutant enzyme Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 8.2
-
 phosphate mutant enzyme Q323A/M324A, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 14
-
 phosphate wild type enzyme, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 18
-
 phosphate mutant enzyme D272A/R274A/Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 166
-
 acetate wild type enzyme, at pH 5.5 and 45°C Entamoeba histolytica
2.7.2.12 372
-
 acetate mutant enzyme Q323G/M324I, at pH 5.5 and 45°C Entamoeba histolytica
2.7.2.12 566
-
 acetate mutant enzyme Q323A/M324A, at pH 5.5 and 45°C Entamoeba histolytica

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.7.2.1 Mg2+ 5 mM used in assay conditions Methanosarcina thermophila
2.7.2.12 Mg2+ 5 mM used in assay conditions Entamoeba histolytica

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.7.2.1 ADP + acetyl phosphate Methanosarcina thermophila
-
 ATP + acetate
-
 r
2.7.2.1 ATP + acetate Methanosarcina thermophila
-
 ADP + acetyl phosphate
-
 r
2.7.2.12 acetate + diphosphate Entamoeba histolytica
-
 acetyl phosphate + phosphate
-
 r
2.7.2.12 acetyl phosphate + phosphate Entamoeba histolytica
-
 acetate + diphosphate
-
 r

Organism

EC Number Organism UniProt Comment Textmining
2.7.2.1 Methanosarcina thermophila P38502
-
-
2.7.2.12 Entamoeba histolytica C4M1C3
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.7.2.1
-
 Methanosarcina thermophila
2.7.2.12
-
 Entamoeba histolytica

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.7.2.1 ADP + acetyl phosphate
-
 Methanosarcina thermophila ATP + acetate
-
 r
2.7.2.1 ATP + acetate
-
 Methanosarcina thermophila ADP + acetyl phosphate
-
 r
2.7.2.12 acetate + diphosphate
-
 Entamoeba histolytica acetyl phosphate + phosphate
-
 r
2.7.2.12 acetyl phosphate + phosphate
-
 Entamoeba histolytica acetate + diphosphate
-
 r

Synonyms

EC Number Synonyms Comment Organism
2.7.2.1 ACK
-
 Methanosarcina thermophila
2.7.2.12 ACK
-
 Entamoeba histolytica

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.7.2.1 12
-
 ADP mutant enzyme G331Q/I332M, at pH 7.0 and 37°C Methanosarcina thermophila
2.7.2.1 12
-
 acetyl phosphate mutant enzyme G331Q/I332M, at pH 7.0 and 37°C Methanosarcina thermophila
2.7.2.1 14
-
 ATP mutant enzyme G331Q/I332M, at pH 5.5 and 45°C Methanosarcina thermophila
2.7.2.1 17
-
 acetate mutant enzyme G331Q/I332M, at pH 5.5 and 45°C Methanosarcina thermophila
2.7.2.1 711
-
 ATP wild type enzyme, at pH 5.5 and 45°C Methanosarcina thermophila
2.7.2.1 789
-
 acetate wild type enzyme, at pH 5.5 and 45°C Methanosarcina thermophila
2.7.2.1 1550
-
 acetyl phosphate wild type enzyme, at pH 7.0 and 37°C Methanosarcina thermophila
2.7.2.1 1915
-
 ADP wild type enzyme, at pH 7.0 and 37°C Methanosarcina thermophila
2.7.2.12 0.052
-
 acetyl phosphate mutant enzyme D272A/R274A/Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 0.076
-
 phosphate mutant enzyme D272A/R274A/Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 0.36
-
 diphosphate mutant enzyme Q323A/M324A, at pH 5.5 and 45°C Entamoeba histolytica
2.7.2.12 0.45
-
 diphosphate mutant enzyme Q323G/M324I, at pH 5.5 and 45°C Entamoeba histolytica
2.7.2.12 0.51
-
 acetate mutant enzyme Q323A/M324A, at pH 5.5 and 45°C Entamoeba histolytica
2.7.2.12 0.51
-
 acetate mutant enzyme Q323G/M324I, at pH 5.5 and 45°C Entamoeba histolytica
2.7.2.12 0.9
-
 diphosphate wild type enzyme, at pH 5.5 and 45°C Entamoeba histolytica
2.7.2.12 1.4
-
 acetate wild type enzyme, at pH 5.5 and 45°C Entamoeba histolytica
2.7.2.12 2 8 phosphate mutant enzyme Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 14
-
 acetyl phosphate mutant enzyme Q323A/M324A, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 20
-
 phosphate mutant enzyme Q323A/M324A, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 32
-
 acetyl phosphate mutant enzyme Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 196
-
 phosphate wild type enzyme, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 266
-
 acetyl phosphate wild type enzyme, at pH 7.0 and 37°C Entamoeba histolytica

IC50 Value

EC Number IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
2.7.2.1 3.3
-
 wild type enzyme, in the acetate-forming direction, at pH 7.0 and 37°C Methanosarcina thermophila diphosphate
2.7.2.1 4
-
 mutant enzyme G331Q/I332M, in the acetate-forming direction, at pH 7.0 and 37°C Methanosarcina thermophila diphosphate
2.7.2.1 7.5
-
 wild type enzyme, in the acetyl phosphate-forming direction, at pH 5.5 and 45°C Methanosarcina thermophila diphosphate
2.7.2.1 14
-
 mutant enzyme G331Q/I332M, in the acetyl phosphate-forming direction, at pH 5.5 and 45°C Methanosarcina thermophila diphosphate
2.7.2.12 2.4
-
 mutant enzyme D272A/R274A/Q323G/M324I, in the acetate-forming direction, at pH 7.0 and 37°C Entamoeba histolytica ATP
2.7.2.12 4.2
-
 mutant enzyme Q323G/M324I, in the acetate-forming direction, at pH 7.0 and 37°C Entamoeba histolytica ATP
2.7.2.12 4.8
-
 mutant enzyme Q323A/M324A, in the acetate-forming direction, at pH 7.0 and 37°C Entamoeba histolytica ATP
2.7.2.12 6.1
-
 wild type enzyme, in the acetate-forming direction, at pH 7.0 and 37°C Entamoeba histolytica ATP

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.7.2.1 0.057
-
 acetate mutant enzyme G331Q/I332M, at pH 5.5 and 45°C Methanosarcina thermophila
2.7.2.1 1.5
-
 ATP mutant enzyme G331Q/I332M, at pH 5.5 and 45°C Methanosarcina thermophila
2.7.2.1 2
-
 ADP mutant enzyme G331Q/I332M, at pH 7.0 and 37°C Methanosarcina thermophila
2.7.2.1 7.2
-
 acetyl phosphate mutant enzyme G331Q/I332M, at pH 7.0 and 37°C Methanosarcina thermophila
2.7.2.1 39
-
 acetate wild type enzyme, at pH 5.5 and 45°C Methanosarcina thermophila
2.7.2.1 421
-
 ATP wild type enzyme, at pH 5.5 and 45°C Methanosarcina thermophila
2.7.2.1 1073
-
 ADP wild type enzyme, at pH 7.0 and 37°C Methanosarcina thermophila
2.7.2.1 1294
-
 acetyl phosphate wild type enzyme, at pH 7.0 and 37°C Methanosarcina thermophila
2.7.2.12 0.0009
-
 acetate mutant enzyme Q323A/M324A, at pH 5.5 and 45°C Entamoeba histolytica
2.7.2.12 0.0014
-
 acetate mutant enzyme Q323G/M324I, at pH 5.5 and 45°C Entamoeba histolytica
2.7.2.12 0.004
-
 phosphate mutant enzyme D272A/R274A/Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 0.0086
-
 acetate wild type enzyme, at pH 5.5 and 45°C Entamoeba histolytica
2.7.2.12 0.08
-
 acetyl phosphate mutant enzyme D272A/R274A/Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 0.16
-
 diphosphate mutant enzyme Q323A/M324A, at pH 5.5 and 45°C Entamoeba histolytica
2.7.2.12 0.23
-
 diphosphate mutant enzyme Q323G/M324I, at pH 5.5 and 45°C Entamoeba histolytica
2.7.2.12 0.44
-
 diphosphate wild type enzyme, at pH 5.5 and 45°C Entamoeba histolytica
2.7.2.12 2.5
-
 phosphate mutant enzyme Q323A/M324A, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 3.8
-
 phosphate mutant enzyme Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 14
-
 phosphate wild type enzyme, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 42
-
 acetyl phosphate mutant enzyme Q323A/M324A, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 68
-
 acetyl phosphate mutant enzyme Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
2.7.2.12 467
-
 acetyl phosphate wild type enzyme, at pH 7.0 and 37°C Entamoeba histolytica