BRENDA - Enzyme Database

Novel allosteric properties produced by residue substitutions in the subunit interface of yeast NAD+-specific isocitrate dehydrogenase

Hu, G.; McAlister-Henn, L.; Arch. Biochem. Biophys. 453, 207-216 (2006)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
1.1.1.41
wild-type and mutant enzymes are expressed in a idh1DELTAidh2DELTA yeast strain which contains deletion/insertion mutations in genomic IDH1 and IDH2 loci; wild-type and mutant enzymes are expressed in a idh1DELTAidh2DELTA yeast strain which contains deletion/insertion mutations in genomic IDH1 and IDH2 loci
Saccharomyces cerevisiae
Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.41
I221A
residue changes in IDH2 subunits
Saccharomyces cerevisiae
1.1.1.41
I221A/V225A/V229A
mutant enzyme IDH1-IDH2(I221A/V225A/V229A) shows an about 6fold decrease in apparent affinity for isocitrate measured in the absence or presence of AMP as compared to wild-type enzyme. Mutant enzyme IDH1-IDH2 (I221A/V225A/V229A) exhibits a moderate decrease in apparent affnity for cofactor (about 2.5fold relative to wild-type) in the absence of AMP. The mutant enzyme has acquired the novel property of cooperativity with respect to NAD+, but this cooperativity is not apparent in the presence of AMP. The mutant enzyme with residue changes in only one subunit are active in vivo; mutant enzyme IDH1-IDH2(I221A/V225A/V229A) shows an about 6fold decrease in apparent affinity for isocitrate measured in the absence or presence of AMP as compared to wild-type enzyme. Mutant enzyme IDH1-IDH2 (I221A/V225A/V229A) exhibits a moderate decrease in apparent affnity for cofactor (about 2.5fold relative to wild-type) in the absence of AMP. The mutant enzyme has acquired the novel property of cooperativity with respect to NAD+, but this cooperativity is not apparent in the presence of AMP. The mutant enzyme with residue changes in only one subunit are active in vivo
Saccharomyces cerevisiae
1.1.1.41
S220A
residue changes in IDH1 subunits
Saccharomyces cerevisiae
1.1.1.41
V214A
residue changes in IDH1 subunits
Saccharomyces cerevisiae
1.1.1.41
V216A/S220A/V224A
IDH1(V216A/S220A/V224A)IDH2 mutant enzyme shows an about 6fold decrease in apparent affinity for isocitrate measured in the absence or presence of AMP as compared to wild-type enzyme. IDH1(V216A/S220A/V224A)IDH2 mutant enzyme exhibits a moderate decrease in apparent affnity for cofactor (about 2.5fold relative to wild-type) in the absence of AMP. The mutant enzyme has acquired the novel property of cooperativity with respect to NAD+, but this cooperativity is not apparent in the presence of AMP. The mutant enzyme with residue changes in only one subunit are active in vivo; IDH1(V216A/S220A/V224A)IDH2 mutant enzyme shows an about 6fold decrease in apparent affinity for isocitrate measured in the absence or presence of AMP as compared to wild-type enzyme. IDH1(V216A/S220A/V224A)IDH2 mutant enzyme exhibits a moderate decrease in apparent affnity for cofactor (about 2.5fold relative to wild-type) in the absence of AMP. The mutant enzyme has acquired the novel property of cooperativity with respect to NAD+, but this cooperativity is not apparent in the presence of AMP. The mutant enzyme with residue changes in only one subunit are active in vivo
Saccharomyces cerevisiae
1.1.1.41
V216A/S220A/V224A/I221A/V225A/V229A
in the absence of AMP, the IDH1(V216A/S220A/V224A)-IDH2(I221A/V225A/V229A) mutant enzyme demonstrates an about 30fold decrease in apparent Vmax relative to wild-type and a loss of cooperativity with respect to isocitrate and, in the presence of AMP, the apparent affinity of the enzyme for isocitrate is 35fold lower than that of the wild-type enzyme. This mutant enzyme also exhibits a significant decrease in apparent affinity for NAD+ (about 30fold in the absence of AMP and about 10fold in the presence of AMP relative to wild-type). The mutant enzyme has acquired the novel property of cooperativity with respect to NAD+, but this cooperativity is not apparent in the presence of AMP. The mutant enzyme demonstrates decreases in apparent affnity for isocitrate of about 12fold in the absence of AMP and of about 26fold in the presence of AMP relative to wild-type. For this mutant enzyme, with respect to isocitrate, allosteric activation by AMP and cooperativity in the absence of AMP are no longer apparen. The mutant enzyme is not fully functional in vivo; in the absence of AMP, the IDH1(V216A/S220A/V224A)-IDH2(I221A/V225A/V229A) mutant enzyme demonstrates an about 30fold decrease in apparent Vmax relative to wild-type and a loss of cooperativity with respect to isocitrate and, in the presence of AMP, the apparent affinity of the enzyme for isocitrate is 35fold lower than that of the wild-type enzyme. This mutant enzyme also exhibits a significant decrease in apparent affinity for NAD+ (about 30fold in the absence of AMP and about 10fold in the presence of AMP relative to wild-type). The mutant enzyme has acquired the novel property of cooperativity with respect to NAD+, but this cooperativity is not apparent in the presence of AMP. The mutant enzyme demonstrates decreases in apparent affnity for isocitrate of about 12fold in the absence of AMP and of about 26fold in the presence of AMP relative to wild-type. For this mutant enzyme, with respect to isocitrate, allosteric activation by AMP and cooperativity in the absence of AMP are no longer apparen. The mutant enzyme is not fully functional in vivo
Saccharomyces cerevisiae
1.1.1.41
V224A
residue changes in IDH1 subunits
Saccharomyces cerevisiae
1.1.1.41
V225A
residue changes in IDH2 subunits
Saccharomyces cerevisiae
1.1.1.41
V229A
residue changes in IDH2 subunits
Saccharomyces cerevisiae
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.1.1.41
37755
-
4 * 38001, 4 * 37755, the enzyme consists of four IDH1 and four IDH2 subunits, the basic structural unit of the enzyme is an IDH1/IDH2 heterodimer
Saccharomyces cerevisiae
1.1.1.41
38001
-
4 * 38001, 4 * 37755, the enzyme consists of four IDH1 and four IDH2 subunits, the basic structural unit of the enzyme is an IDH1/IDH2 heterodimer
Saccharomyces cerevisiae
1.1.1.41
335000
-
gel filtration; wild-type enzyme, gel filtration; wild-type enzyme, gel filtration
Saccharomyces cerevisiae
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.1.1.41
Saccharomyces cerevisiae
P28241
IDH2
-
1.1.1.41
Saccharomyces cerevisiae
P28834
IDH1
-
1.1.1.41
Saccharomyces cerevisiae
-
-
-
1.1.1.41
Saccharomyces cerevisiae MMY011
-
-
-
Purification (Commentary)
EC Number
Commentary
Organism
1.1.1.41
Ni2+-nitrilotriacetate (Ni2+-NTA) column chromatography
Saccharomyces cerevisiae
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.1.1.41
D-isocitrate + NAD+
-
667332
Saccharomyces cerevisiae
2-oxoglutarate + CO2 + NADH
-
-
-
ir
1.1.1.41
D-isocitrate + NAD+
-
667332
Saccharomyces cerevisiae MMY011
2-oxoglutarate + CO2 + NADH
-
-
-
ir
1.1.1.41
isocitrate + NAD+
-
667332
Saccharomyces cerevisiae
2-oxoglutarate + CO2 + NADH + H+
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
1.1.1.41
octamer
4 * 38001, 4 * 37755, the enzyme consists of four IDH1 and four IDH2 subunits, the basic structural unit of the enzyme is an IDH1/IDH2 heterodimer; 4 * IDH1 + 4 * IDH2; 4 * IDH1 + 4 * IDH2
Saccharomyces cerevisiae
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.41
wild-type and mutant enzymes are expressed in a idh1DELTAidh2DELTA yeast strain which contains deletion/insertion mutations in genomic IDH1 and IDH2 loci
Saccharomyces cerevisiae
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.41
I221A
residue changes in IDH2 subunits
Saccharomyces cerevisiae
1.1.1.41
I221A/V225A/V229A
mutant enzyme IDH1-IDH2(I221A/V225A/V229A) shows an about 6fold decrease in apparent affinity for isocitrate measured in the absence or presence of AMP as compared to wild-type enzyme. Mutant enzyme IDH1-IDH2 (I221A/V225A/V229A) exhibits a moderate decrease in apparent affnity for cofactor (about 2.5fold relative to wild-type) in the absence of AMP. The mutant enzyme has acquired the novel property of cooperativity with respect to NAD+, but this cooperativity is not apparent in the presence of AMP. The mutant enzyme with residue changes in only one subunit are active in vivo
Saccharomyces cerevisiae
1.1.1.41
S220A
residue changes in IDH1 subunits
Saccharomyces cerevisiae
1.1.1.41
V214A
residue changes in IDH1 subunits
Saccharomyces cerevisiae
1.1.1.41
V216A/S220A/V224A
IDH1(V216A/S220A/V224A)IDH2 mutant enzyme shows an about 6fold decrease in apparent affinity for isocitrate measured in the absence or presence of AMP as compared to wild-type enzyme. IDH1(V216A/S220A/V224A)IDH2 mutant enzyme exhibits a moderate decrease in apparent affnity for cofactor (about 2.5fold relative to wild-type) in the absence of AMP. The mutant enzyme has acquired the novel property of cooperativity with respect to NAD+, but this cooperativity is not apparent in the presence of AMP. The mutant enzyme with residue changes in only one subunit are active in vivo
Saccharomyces cerevisiae
1.1.1.41
V216A/S220A/V224A/I221A/V225A/V229A
in the absence of AMP, the IDH1(V216A/S220A/V224A)-IDH2(I221A/V225A/V229A) mutant enzyme demonstrates an about 30fold decrease in apparent Vmax relative to wild-type and a loss of cooperativity with respect to isocitrate and, in the presence of AMP, the apparent affinity of the enzyme for isocitrate is 35fold lower than that of the wild-type enzyme. This mutant enzyme also exhibits a significant decrease in apparent affinity for NAD+ (about 30fold in the absence of AMP and about 10fold in the presence of AMP relative to wild-type). The mutant enzyme has acquired the novel property of cooperativity with respect to NAD+, but this cooperativity is not apparent in the presence of AMP. The mutant enzyme demonstrates decreases in apparent affnity for isocitrate of about 12fold in the absence of AMP and of about 26fold in the presence of AMP relative to wild-type. For this mutant enzyme, with respect to isocitrate, allosteric activation by AMP and cooperativity in the absence of AMP are no longer apparen. The mutant enzyme is not fully functional in vivo
Saccharomyces cerevisiae
1.1.1.41
V224A
residue changes in IDH1 subunits
Saccharomyces cerevisiae
1.1.1.41
V225A
residue changes in IDH2 subunits
Saccharomyces cerevisiae
1.1.1.41
V229A
residue changes in IDH2 subunits
Saccharomyces cerevisiae
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.1.1.41
37755
-
4 * 38001, 4 * 37755, the enzyme consists of four IDH1 and four IDH2 subunits, the basic structural unit of the enzyme is an IDH1/IDH2 heterodimer
Saccharomyces cerevisiae
1.1.1.41
38001
-
4 * 38001, 4 * 37755, the enzyme consists of four IDH1 and four IDH2 subunits, the basic structural unit of the enzyme is an IDH1/IDH2 heterodimer
Saccharomyces cerevisiae
1.1.1.41
335000
-
gel filtration
Saccharomyces cerevisiae
1.1.1.41
335000
-
wild-type enzyme, gel filtration
Saccharomyces cerevisiae
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.41
-
Saccharomyces cerevisiae
1.1.1.41
Ni2+-nitrilotriacetate (Ni2+-NTA) column chromatography
Saccharomyces cerevisiae
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.1.1.41
D-isocitrate + NAD+
-
667332
Saccharomyces cerevisiae
2-oxoglutarate + CO2 + NADH
-
-
-
ir
1.1.1.41
D-isocitrate + NAD+
-
667332
Saccharomyces cerevisiae MMY011
2-oxoglutarate + CO2 + NADH
-
-
-
ir
1.1.1.41
isocitrate + NAD+
-
667332
Saccharomyces cerevisiae
2-oxoglutarate + CO2 + NADH + H+
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
1.1.1.41
octamer
4 * 38001, 4 * 37755, the enzyme consists of four IDH1 and four IDH2 subunits, the basic structural unit of the enzyme is an IDH1/IDH2 heterodimer
Saccharomyces cerevisiae
1.1.1.41
octamer
4 * IDH1 + 4 * IDH2
Saccharomyces cerevisiae