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Literature summary for 2.7.2.12 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
Search for more literature for 2.7.2.12

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli YBS121 DELTAack DELTApta cells Entamoeba histolytica

Protein Variants

Protein Variants Comment Organism
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
DELTAG203/Q323G/M324I inactive Entamoeba histolytica
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
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

Inhibitors Comment Organism Structure
ADP 80% inhibition at 1 mM Entamoeba histolytica
ATP about 30% inhibition at 1 mM Entamoeba histolytica
additional information not inhibited by AMP Entamoeba histolytica

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.34
-
 acetyl phosphate mutant enzyme Q323A/M324A, at pH 7.0 and 37°C Entamoeba histolytica
0.47
-
 acetyl phosphate mutant enzyme Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
0.57
-
 acetyl phosphate wild type enzyme, at pH 7.0 and 37°C Entamoeba histolytica
0.66
-
 acetyl phosphate mutant enzyme D272A/R274A/Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
2
-
 diphosphate mutant enzyme Q323G/M324I, at pH 5.5 and 45°C Entamoeba histolytica
2.1
-
 diphosphate wild type enzyme, at pH 5.5 and 45°C Entamoeba histolytica
2.3
-
 diphosphate mutant enzyme Q323A/M324A, at pH 5.5 and 45°C Entamoeba histolytica
7.3
-
 phosphate mutant enzyme Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
8.2
-
 phosphate mutant enzyme Q323A/M324A, at pH 7.0 and 37°C Entamoeba histolytica
14
-
 phosphate wild type enzyme, at pH 7.0 and 37°C Entamoeba histolytica
18
-
 phosphate mutant enzyme D272A/R274A/Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
166
-
 acetate wild type enzyme, at pH 5.5 and 45°C Entamoeba histolytica
372
-
 acetate mutant enzyme Q323G/M324I, at pH 5.5 and 45°C Entamoeba histolytica
566
-
 acetate mutant enzyme Q323A/M324A, at pH 5.5 and 45°C Entamoeba histolytica

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ 5 mM used in assay conditions Entamoeba histolytica

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
acetate + diphosphate Entamoeba histolytica
-
 acetyl phosphate + phosphate
-
 r
acetyl phosphate + phosphate Entamoeba histolytica
-
 acetate + diphosphate
-
 r

Organism

Organism UniProt Comment Textmining
Entamoeba histolytica C4M1C3
-
-

Purification (Commentary)

Purification (Comment) Organism
-
 Entamoeba histolytica

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
acetate + diphosphate
-
 Entamoeba histolytica acetyl phosphate + phosphate
-
 r
acetyl phosphate + phosphate
-
 Entamoeba histolytica acetate + diphosphate
-
 r

Synonyms

Synonyms Comment Organism
ACK
-
 Entamoeba histolytica

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.052
-
 acetyl phosphate mutant enzyme D272A/R274A/Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
0.076
-
 phosphate mutant enzyme D272A/R274A/Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
0.36
-
 diphosphate mutant enzyme Q323A/M324A, at pH 5.5 and 45°C Entamoeba histolytica
0.45
-
 diphosphate mutant enzyme Q323G/M324I, at pH 5.5 and 45°C Entamoeba histolytica
0.51
-
 acetate mutant enzyme Q323A/M324A, at pH 5.5 and 45°C Entamoeba histolytica
0.51
-
 acetate mutant enzyme Q323G/M324I, at pH 5.5 and 45°C Entamoeba histolytica
0.9
-
 diphosphate wild type enzyme, at pH 5.5 and 45°C Entamoeba histolytica
1.4
-
 acetate wild type enzyme, at pH 5.5 and 45°C Entamoeba histolytica
2 8 phosphate mutant enzyme Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
14
-
 acetyl phosphate mutant enzyme Q323A/M324A, at pH 7.0 and 37°C Entamoeba histolytica
20
-
 phosphate mutant enzyme Q323A/M324A, at pH 7.0 and 37°C Entamoeba histolytica
32
-
 acetyl phosphate mutant enzyme Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
196
-
 phosphate wild type enzyme, at pH 7.0 and 37°C Entamoeba histolytica
266
-
 acetyl phosphate wild type enzyme, at pH 7.0 and 37°C Entamoeba histolytica

IC50 Value

IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
2.4
-
 mutant enzyme D272A/R274A/Q323G/M324I, in the acetate-forming direction, at pH 7.0 and 37°C Entamoeba histolytica ATP
4.2
-
 mutant enzyme Q323G/M324I, in the acetate-forming direction, at pH 7.0 and 37°C Entamoeba histolytica ATP
4.8
-
 mutant enzyme Q323A/M324A, in the acetate-forming direction, at pH 7.0 and 37°C Entamoeba histolytica ATP
6.1
-
 wild type enzyme, in the acetate-forming direction, at pH 7.0 and 37°C Entamoeba histolytica ATP

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.0009
-
 acetate mutant enzyme Q323A/M324A, at pH 5.5 and 45°C Entamoeba histolytica
0.0014
-
 acetate mutant enzyme Q323G/M324I, at pH 5.5 and 45°C Entamoeba histolytica
0.004
-
 phosphate mutant enzyme D272A/R274A/Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
0.0086
-
 acetate wild type enzyme, at pH 5.5 and 45°C Entamoeba histolytica
0.08
-
 acetyl phosphate mutant enzyme D272A/R274A/Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
0.16
-
 diphosphate mutant enzyme Q323A/M324A, at pH 5.5 and 45°C Entamoeba histolytica
0.23
-
 diphosphate mutant enzyme Q323G/M324I, at pH 5.5 and 45°C Entamoeba histolytica
0.44
-
 diphosphate wild type enzyme, at pH 5.5 and 45°C Entamoeba histolytica
2.5
-
 phosphate mutant enzyme Q323A/M324A, at pH 7.0 and 37°C Entamoeba histolytica
3.8
-
 phosphate mutant enzyme Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
14
-
 phosphate wild type enzyme, at pH 7.0 and 37°C Entamoeba histolytica
42
-
 acetyl phosphate mutant enzyme Q323A/M324A, at pH 7.0 and 37°C Entamoeba histolytica
68
-
 acetyl phosphate mutant enzyme Q323G/M324I, at pH 7.0 and 37°C Entamoeba histolytica
467
-
 acetyl phosphate wild type enzyme, at pH 7.0 and 37°C Entamoeba histolytica