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Literature summary for 4.1.3.1 extracted from

  • Moynihan, M.M.; Murkin, A.S.
    Cysteine is the general base that serves in catalysis by isocitrate lyase and in mechanism-based inhibition by 3-nitropropionate (2014), Biochemistry, 53, 178-187.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
expression of the enzyme with a thrombin-cleavable N-terminal His6 tag in Escherichia coli strain BL21(DE3) Mycobacterium tuberculosis

Inhibitors

Inhibitors Comment Organism Structure
3-nitropropionate a succinate analogue. Inhibition by 3-nitropropionate proceeds through an unusual double slow-onset process featuring formation of a complex with a Ki of 0.0033 mM during the first minute, followed by formation of a final complex with a Ki* of 44 nM over the course of several min to hours Mycobacterium tuberculosis
glyoxylate
-
Mycobacterium tuberculosis
Itaconate a succinate analogue lacking an acidic alpha-proton, the inhhibitor does not display slow-binding behavior Mycobacterium tuberculosis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information stopped-flow spectroscopy and steady-state kinetics, solvent kinetic isotope effects, overview. Whereas the D2OV is consistent with partially rate-limiting proton transfer during formation of the hydroxyl group of isocitrate, the large inverse D2O(V/Ksuccinate) indicates that substantially different kinetic parameters exist when the enzyme is saturated with succinate Mycobacterium tuberculosis
0.045
-
isocitrate recombinant detagged enzyme, pH 7.0, 37°C Mycobacterium tuberculosis
0.14
-
glyoxylate recombinant detagged enzyme, pH 7.0, 37°C Mycobacterium tuberculosis
0.412
-
succinate recombinant detagged enzyme, pH 7.0, 37°C Mycobacterium tuberculosis

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Mycobacterium tuberculosis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
isocitrate Mycobacterium tuberculosis
-
succinate + glyoxylate
-
r
isocitrate Mycobacterium tuberculosis H37R
-
succinate + glyoxylate
-
r

Organism

Organism UniProt Comment Textmining
Mycobacterium tuberculosis
-
-
-
Mycobacterium tuberculosis H37R
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant N-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nicke affinity chromatography, cleavage of the His-tag by thrombin, and dialysis Mycobacterium tuberculosis

Reaction

Reaction Comment Organism Reaction ID
isocitrate = succinate + glyoxylate common mechanism for catalysis with succinate: (1) an unfavorable prebinding isomerization of the active site Cys191-His193 pair to the thiolate-imidazolium form, a process that is favored in D2O, and (2) the transfer of a proton from succinate or 3-NP to Cys191 Mycobacterium tuberculosis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
isocitrate
-
Mycobacterium tuberculosis succinate + glyoxylate
-
r
isocitrate
-
Mycobacterium tuberculosis H37R succinate + glyoxylate
-
r
succinate + glyoxylate
-
Mycobacterium tuberculosis isocitrate
-
r
succinate + glyoxylate
-
Mycobacterium tuberculosis H37R isocitrate
-
r

Synonyms

Synonyms Comment Organism
ICL
-
Mycobacterium tuberculosis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
assay at Mycobacterium tuberculosis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
8.5
-
succinate recombinant detagged enzyme, pH 7.0, 37°C Mycobacterium tuberculosis
12.2
-
isocitrate recombinant detagged enzyme, pH 7.0, 37°C Mycobacterium tuberculosis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
assay at Mycobacterium tuberculosis

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
additional information
-
additional information solvent isotope effects in inhibition by itaconate and 3-nitropropionate, overview Mycobacterium tuberculosis
4.3
-
glyoxylate recombinant detagged enzyme, pH 7.0, 37°C Mycobacterium tuberculosis

General Information

General Information Comment Organism
physiological function the enzyme catalyzes the first committed step in the glyoxylate cycle, it is essential in Mycobacterium tuberculosis for cell survival during chronic infection Mycobacterium tuberculosis