Application | Comment | Organism |
---|---|---|
medicine | hereditary SCOT deficiency is one cause of ketoacidosis | Sus scrofa |
Cloned (Comment) | Organism |
---|---|
expression in Escherichia coli BL21-DE3 | Sus scrofa |
expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Sus scrofa |
Crystallization (Comment) | Organism |
---|---|
14°C, hanging drop vapor diffusion, structure determination with X-ray crystallography | Sus scrofa |
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 | Comment | Organism |
---|---|---|
C129S | like wild-type SCOT rapid modification in the presence of acyl-CoA substrates | Sus scrofa |
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 |
C196S | specific activity similar to wild-type SCOT | Sus scrofa |
C28A | specific activity similar to C28S mutant | Sus scrofa |
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 |
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 | Comment | Organism | Structure |
---|---|---|---|
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 | |
acetoacetate | product inhibition | Sus scrofa | |
Br- | same inhibition as I-, stronger inhibition than Cl-, the kind of cation has no inhibitory effect | Sus scrofa | |
Cl- | lower inhibition than Br-, stronger inhibition than F-, the kind of cation has no inhibitory effect | Sus scrofa | |
ClO4- | lower inhibition than SCN-, stronger inhibition than I-, the kind of cation has no inhibitory effect | Sus scrofa | |
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 | |
F- | lowest inhibition, the kind of cation has no inhibitory effect | Sus scrofa | |
I- | lower inhibition than ClO4-, same inhibition as Br-, the kind of cation has no inhibitory effect | Sus scrofa | |
additional information | the DNA region between -2168 and -361 appears to inhibit the SCOT promoter activity in HepG2 cells | Sus scrofa | |
SCN- | strongest inhibition, the kind of cation has no inhibitory effect | Sus scrofa | |
succinyl-CoA | product inhibition | Sus scrofa |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.05 | - |
acetoacetate | pH 8.1, 21-22°C, mutant C28S | Sus scrofa | |
0.05 | - |
acetoacetate | C28S mutant | Sus scrofa | |
0.06 | - |
acetoacetyl-CoA | pH 8.1, 21-22°C, mutant C196S | Sus scrofa | |
0.06 | - |
acetoacetyl-CoA | C28A mutant | Sus scrofa | |
0.1 | - |
acetoacetate | pH 8.1, 21-22°C, mutant C196S | Sus scrofa | |
0.1 | - |
acetoacetyl-CoA | pH 8.1, 21-22°C, mutant C28S | Sus scrofa | |
0.1 | - |
acetoacetate | pH 8.1, 21-22°C, wild-type enzyme | Sus scrofa | |
0.1 | - |
acetoacetate | C28A mutant | Sus scrofa | |
0.1 | - |
acetoacetyl-CoA | C28S mutant | Sus scrofa | |
0.1 | - |
acetoacetate | wild-type SCOT | Sus scrofa | |
0.2 | - |
acetoacetyl-CoA | pH 8.1, 21-22°C, wild-type enzyme | Sus scrofa | |
0.2 | - |
acetoacetyl-CoA | wild-type SCOT | Sus scrofa | |
1.7 | - |
succinyl-CoA | pH 8.1, 21-22°C, mutant C28S | Sus scrofa | |
1.7 | - |
succinyl-CoA | C28S mutant | Sus scrofa | |
6.5 | - |
succinyl-CoA | pH 8.1, 21-22°C, wild-type enzyme | Sus scrofa | |
6.5 | - |
succinyl-CoA | wild-type SCOT | Sus scrofa | |
8 | - |
succinyl-CoA | pH 8.1, 21-22°C, mutant C196S | Sus scrofa | |
8 | - |
succinyl-CoA | C28A mutant | Sus scrofa | |
13 | - |
succinate | pH 8.1, 21-22°C, mutant C196S | Sus scrofa | |
13 | - |
succinate | C28A mutant | Sus scrofa | |
20 | - |
succinate | pH 8.1, 21-22°C, wild-type enzyme | Sus scrofa | |
20 | - |
succinate | wild-type SCOT | Sus scrofa | |
39 | - |
succinate | pH 8.1, 21-22°C, mutant C28S | Sus scrofa | |
39 | - |
succinate | C28S mutant | Sus scrofa |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
mitochondrion | - |
Sus scrofa | 5739 | - |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | - |
Sus scrofa |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
succinyl-CoA + a 3-oxo acid | Sus scrofa | - |
succinate + a 3-oxoacyl-CoA | - |
? | |
succinyl-CoA + acetoacetate | Sus scrofa | - |
succinate + acetoacetyl-CoA | - |
r | |
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 | |
succinyl-CoA + beta-hydroxybutyrate | Sus scrofa | essential enzyme in the metabolism of ketone bodies in higher animals | succinate + beta-hydroxybutyryl-CoA | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Sus scrofa | - |
- |
- |
Sus scrofa | Q29551 | - |
- |
Purification (Comment) | Organism |
---|---|
as described in literature | Sus scrofa |
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) to homogeneity | Sus scrofa |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
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 | Comment | Organism | Textmining |
---|---|---|---|
heart | - |
Sus scrofa | - |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
6 | - |
C28A mutant, similar to C28S mutant | Sus scrofa |
7.1 | - |
purified recombinant mutant C28S | Sus scrofa |
7.1 | - |
C28S mutant, 3.6times lower than wild-type enzyme | Sus scrofa |
22.4 | - |
purified recombinant mutant C196S | Sus scrofa |
22.4 | - |
C196S mutant | Sus scrofa |
25.2 | - |
purified recombinant wild-type enzyme | Sus scrofa |
25.2 | - |
wild-type SCOT | Sus scrofa |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
succinyl-CoA + a 3-oxo acid | - |
Sus scrofa | succinate + a 3-oxoacyl-CoA | - |
? | |
succinyl-CoA + acetoacetate | - |
Sus scrofa | succinate + acetoacetyl-CoA | - |
r | |
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 | |
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 | |
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 | |
succinyl-CoA + beta-hydroxybutyrate | essential enzyme in the metabolism of ketone bodies in higher animals | Sus scrofa | succinate + beta-hydroxybutyryl-CoA | - |
? |
Subunits | Comment | Organism |
---|---|---|
homodimer | extracted from literature | Sus scrofa |
homotetramer | extracted from literature, homotetramer has an identical specific activity to the homodimer | Sus scrofa |
Synonyms | Comment | Organism |
---|---|---|
SCOT | - |
Sus scrofa |
succinyl-CoA:3-ketoacid coenzyme A transferase | - |
Sus scrofa |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
21 | 22 | assay at | Sus scrofa |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
9.17 | - |
acetoacetate | pH 8.1, 21-22°C, mutant C28S | Sus scrofa | |
35 | - |
acetoacetate | pH 8.1, 21-22°C, mutant C196S | Sus scrofa | |
58.3 | - |
acetoacetyl-CoA | pH 8.1, 21-22°C, mutant C196S | Sus scrofa | |
98.3 | - |
acetoacetate | pH 8.1, 21-22°C, wild-type enzyme | Sus scrofa | |
816.7 | - |
acetoacetyl-CoA | pH 8.1, 21-22°C, mutant C28S | Sus scrofa | |
1250 | - |
acetoacetyl-CoA | pH 8.1, 21-22°C, wild-type enzyme | Sus scrofa |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8.1 | 9.1 | assay at | Sus scrofa |