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Literature summary extracted from

  • Tammam, S.D.; Rochet, J.C.; Fraser, M.E.
    Identification of the cysteine residue exposed by the conformational change in pig heart succinyl-CoA:3-ketoacid coenzyme A transferase on binding coenzyme A (2007), Biochemistry, 46, 10852-10863.
    View publication on PubMed

Application

EC Number Application Comment Organism
2.8.3.5 medicine hereditary SCOT deficiency is one cause of ketoacidosis Sus scrofa

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.8.3.5 expression in Escherichia coli BL21-DE3 Sus scrofa
2.8.3.5 expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Sus scrofa

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.8.3.5 14°C, hanging drop vapor diffusion, structure determination with X-ray crystallography Sus scrofa
2.8.3.5 purified recombinant C28S and C28A mutants, hanging drop vapour diffusion method, enzyme in 0.125-0.5 M KCl, 0.5 mM benzamidine, 0.2 mM EDTA, 20 mM 2-mercaptoethanol, and 5 mM Tris-HCl, pH 8.0, the precipitant solution contains 14-22% polyethylene glycol 2000, 0.1 M Tris-HCl, pH 8.0, and 10-15% glycerol, X-ray diffraction structure determination and analysis at 2.0-2.05 A resolution Sus scrofa

Protein Variants

EC Number Protein Variants Comment Organism
2.8.3.5 C129S like wild-type SCOT rapid modification in the presence of acyl-CoA substrates Sus scrofa
2.8.3.5 C196S site-directed mutagenesis, the mutant protein is modified rapidly in the presence of acyl-CoA substrates in analogy to the wild-type enzyme Sus scrofa
2.8.3.5 C196S specific activity similar to wild-type SCOT Sus scrofa
2.8.3.5 C28A specific activity similar to C28S mutant Sus scrofa
2.8.3.5 C28S site-directed mutagenesis, the mutant protein is modified much more slowly than the wild-type enzyme, indicating that Cys28 is the residue exposed on binding CoA, the specific activity of the C28S mutant protein is unexpectedly lower than that of wild-type SCOT Sus scrofa
2.8.3.5 C28S slow modification in the presence of acyl-CoA substrates, a chloride ion is bound to one of four active sites in the crystal structure of the C28S mutant protein, mimicking substrate, interacting with Lys329, Asn51, and Asn52 Sus scrofa

Inhibitors

EC Number Inhibitors Comment Organism Structure
2.8.3.5 5,5'-dithiobis(2-nitrobenzoic acid) slow inactivation of the free enzyme by the thiol-modifying reagent DTNB, wild-type SCOT and C196S are both inactivated by DTNB with more rapid kinetics in the presence of succinyl- or acetoacetyl-CoA (about 70-80% inactivation in 25 min) than in the absence of acyl-CoA (only about 30% inactivation in 25 min and about 40-50% inactivation in 50 min), the rate of C28S inactivation by DTNB is accelerated to a much lesser extent by succinyl- or acetoacetyl-CoA, and C28S is inactivated less rapidly than wild-type SCOT or C196S, the inactivation with DNTB is reversible, the enzyme can be reactivated by adding dithiothreitol, kinetics of inactivation by DNTB Sus scrofa
2.8.3.5 acetoacetate product inhibition Sus scrofa
2.8.3.5 Br- same inhibition as I-, stronger inhibition than Cl-, the kind of cation has no inhibitory effect Sus scrofa
2.8.3.5 Cl- lower inhibition than Br-, stronger inhibition than F-, the kind of cation has no inhibitory effect Sus scrofa
2.8.3.5 ClO4- lower inhibition than SCN-, stronger inhibition than I-, the kind of cation has no inhibitory effect Sus scrofa
2.8.3.5 DTNB rapid inactivation of wild-type enzyme and mutant C196S, less rapid inactivation of mutant C28S, CoA, linked to the enzyme, allows the rapid modification of Cys28 by 5,5'-dithiobis(2-nitrobenzoicacid), reflecting a conformational change of SCOT upon formation of the thioester, overview Sus scrofa
2.8.3.5 F- lowest inhibition, the kind of cation has no inhibitory effect Sus scrofa
2.8.3.5 I- lower inhibition than ClO4-, same inhibition as Br-, the kind of cation has no inhibitory effect Sus scrofa
2.8.3.5 additional information the DNA region between -2168 and -361 appears to inhibit the SCOT promoter activity in HepG2 cells Sus scrofa
2.8.3.5 SCN- strongest inhibition, the kind of cation has no inhibitory effect Sus scrofa
2.8.3.5 succinyl-CoA product inhibition Sus scrofa

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.8.3.5 0.05
-
acetoacetate pH 8.1, 21-22°C, mutant C28S Sus scrofa
2.8.3.5 0.05
-
acetoacetate C28S mutant Sus scrofa
2.8.3.5 0.06
-
acetoacetyl-CoA pH 8.1, 21-22°C, mutant C196S Sus scrofa
2.8.3.5 0.06
-
acetoacetyl-CoA C28A mutant Sus scrofa
2.8.3.5 0.1
-
acetoacetate pH 8.1, 21-22°C, mutant C196S Sus scrofa
2.8.3.5 0.1
-
acetoacetyl-CoA pH 8.1, 21-22°C, mutant C28S Sus scrofa
2.8.3.5 0.1
-
acetoacetate pH 8.1, 21-22°C, wild-type enzyme Sus scrofa
2.8.3.5 0.1
-
acetoacetate C28A mutant Sus scrofa
2.8.3.5 0.1
-
acetoacetyl-CoA C28S mutant Sus scrofa
2.8.3.5 0.1
-
acetoacetate wild-type SCOT Sus scrofa
2.8.3.5 0.2
-
acetoacetyl-CoA pH 8.1, 21-22°C, wild-type enzyme Sus scrofa
2.8.3.5 0.2
-
acetoacetyl-CoA wild-type SCOT Sus scrofa
2.8.3.5 1.7
-
succinyl-CoA pH 8.1, 21-22°C, mutant C28S Sus scrofa
2.8.3.5 1.7
-
succinyl-CoA C28S mutant Sus scrofa
2.8.3.5 6.5
-
succinyl-CoA pH 8.1, 21-22°C, wild-type enzyme Sus scrofa
2.8.3.5 6.5
-
succinyl-CoA wild-type SCOT Sus scrofa
2.8.3.5 8
-
succinyl-CoA pH 8.1, 21-22°C, mutant C196S Sus scrofa
2.8.3.5 8
-
succinyl-CoA C28A mutant Sus scrofa
2.8.3.5 13
-
succinate pH 8.1, 21-22°C, mutant C196S Sus scrofa
2.8.3.5 13
-
succinate C28A mutant Sus scrofa
2.8.3.5 20
-
succinate pH 8.1, 21-22°C, wild-type enzyme Sus scrofa
2.8.3.5 20
-
succinate wild-type SCOT Sus scrofa
2.8.3.5 39
-
succinate pH 8.1, 21-22°C, mutant C28S Sus scrofa
2.8.3.5 39
-
succinate C28S mutant Sus scrofa

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
2.8.3.5 mitochondrion
-
Sus scrofa 5739
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.8.3.5 Mg2+
-
Sus scrofa

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.8.3.5 succinyl-CoA + a 3-oxo acid Sus scrofa
-
succinate + a 3-oxoacyl-CoA
-
?
2.8.3.5 succinyl-CoA + acetoacetate Sus scrofa
-
succinate + acetoacetyl-CoA
-
r
2.8.3.5 succinyl-CoA + acetoacetate Sus scrofa essential enzyme in the metabolism of ketone bodies in higher animals, reaction via an enzyme-thioester intermediate succinate + acetoacetyl-CoA
-
r
2.8.3.5 succinyl-CoA + beta-hydroxybutyrate Sus scrofa essential enzyme in the metabolism of ketone bodies in higher animals succinate + beta-hydroxybutyryl-CoA
-
?

Organism

EC Number Organism UniProt Comment Textmining
2.8.3.5 Sus scrofa
-
-
-
2.8.3.5 Sus scrofa Q29551
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.8.3.5 as described in literature Sus scrofa
2.8.3.5 recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) to homogeneity Sus scrofa

Reaction

EC Number Reaction Comment Organism Reaction ID
2.8.3.5 succinyl-CoA + a 3-oxo acid = succinate + a 3-oxoacyl-CoA catalytic mechanism via a Glu-succinate anhydride, a Glu-CoA thioester, and a Glu-acetoacetate anhydride intermediate, Glu305 is the catalytic residue in the active site, overview Sus scrofa

Source Tissue

EC Number Source Tissue Comment Organism Textmining
2.8.3.5 heart
-
Sus scrofa
-

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.8.3.5 6
-
C28A mutant, similar to C28S mutant Sus scrofa
2.8.3.5 7.1
-
purified recombinant mutant C28S Sus scrofa
2.8.3.5 7.1
-
C28S mutant, 3.6times lower than wild-type enzyme Sus scrofa
2.8.3.5 22.4
-
purified recombinant mutant C196S Sus scrofa
2.8.3.5 22.4
-
C196S mutant Sus scrofa
2.8.3.5 25.2
-
purified recombinant wild-type enzyme Sus scrofa
2.8.3.5 25.2
-
wild-type SCOT Sus scrofa

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.8.3.5 succinyl-CoA + a 3-oxo acid
-
Sus scrofa succinate + a 3-oxoacyl-CoA
-
?
2.8.3.5 succinyl-CoA + acetoacetate
-
Sus scrofa succinate + acetoacetyl-CoA
-
r
2.8.3.5 succinyl-CoA + acetoacetate SCOT transfers CoA from succinyl-CoA to acetoacetate via a thioester intermediate with its active site glutamate residue Glu305 Sus scrofa succinate + acetoacetyl-CoA
-
r
2.8.3.5 succinyl-CoA + acetoacetate essential enzyme in the metabolism of ketone bodies in higher animals, reaction via an enzyme-thioester intermediate Sus scrofa succinate + acetoacetyl-CoA
-
r
2.8.3.5 succinyl-CoA + acetoacetate proposal of a new mechanism for catalysis by SCOT, the experiments contrasting the labeling of the C28S and C196S mutant proteins and wild-type SCOT clearly show that Cys 28 is the cysteine residue exposed on binding CoA in the enzyme-thioester intermediate Sus scrofa succinate + acetoacetyl-CoA
-
r
2.8.3.5 succinyl-CoA + beta-hydroxybutyrate essential enzyme in the metabolism of ketone bodies in higher animals Sus scrofa succinate + beta-hydroxybutyryl-CoA
-
?

Subunits

EC Number Subunits Comment Organism
2.8.3.5 homodimer extracted from literature Sus scrofa
2.8.3.5 homotetramer extracted from literature, homotetramer has an identical specific activity to the homodimer Sus scrofa

Synonyms

EC Number Synonyms Comment Organism
2.8.3.5 SCOT
-
Sus scrofa
2.8.3.5 succinyl-CoA:3-ketoacid coenzyme A transferase
-
Sus scrofa

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.8.3.5 21 22 assay at Sus scrofa

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.8.3.5 9.17
-
acetoacetate pH 8.1, 21-22°C, mutant C28S Sus scrofa
2.8.3.5 35
-
acetoacetate pH 8.1, 21-22°C, mutant C196S Sus scrofa
2.8.3.5 58.3
-
acetoacetyl-CoA pH 8.1, 21-22°C, mutant C196S Sus scrofa
2.8.3.5 98.3
-
acetoacetate pH 8.1, 21-22°C, wild-type enzyme Sus scrofa
2.8.3.5 816.7
-
acetoacetyl-CoA pH 8.1, 21-22°C, mutant C28S Sus scrofa
2.8.3.5 1250
-
acetoacetyl-CoA pH 8.1, 21-22°C, wild-type enzyme Sus scrofa

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.8.3.5 8.1 9.1 assay at Sus scrofa