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A676T
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private non-conservative allele detected in ENPEP, nucleotide change 2028G>A
D221A
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mutant constructed for the examination of Ca2+ effects on enzymatic activity
D221E
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mutant constructed for the examination of Ca2+ effects on enzymatic activity
D221N
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mutant constructed for the examination of Ca2+ effects on enzymatic activity
D221Q
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mutant constructed for the examination of Ca2+ effects on enzymatic activity
D622N
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private non-conservative allele detected in ENPEP, nucleotide change 1866G>A
E172Q
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polymorphism detected in ENPEP, missense mutation, nucleotide change 516G>C
E686K
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private non-conservative allele detected in ENPEP, nucleotide change 2058G>A
E687D
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private non-conservative allele detected in ENPEP, nucleotide change 2061G>T
I32V
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polymorphism detected in ENPEP, missense mutation, nucleotide change 96A>G
K923I
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private non-conservative allele detected in ENPEP, nucleotide change 2769A>T
Q213R
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polymorphism detected in ENPEP, missense mutation, nucleotide change 639A>G
Q435E
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private non-conservative allele detected in ENPEP, nucleotide change 1305C>G
R159S
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polymorphism detected in ENPEP, missense mutation, nucleotide change 477G>T
R925G
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private non-conservative allele detected in ENPEP, nucleotide change 2775A>G
S861R
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polymorphism detected in ENPEP, missense mutation, nucleotide change 2583C>G
V218A
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polymorphism detected in ENPEP, missense mutation, nucleotide change 654A>C
W413X
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polymorphism detected in ENPEP, nonsense mutation, nucleotide change 1239G>T
Y544F
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polymorphism detected in ENPEP, missense mutation, nucleotide change 1632A>T
C43S
mutant enzyme is monomeric under reducing and under non-reducing conditions, specific activity of the mutant enzyme is similar to that of the wild-type enzyme
D227A
mutant enzyme produces only the 140000 Da enzyme form by SDS-PAGE and autoradiography compared to a 168000 Da enzyme form and a 140000 Da enzyme form of the wild-type enzyme. Mutant enzyme displays incorrect maturation and trafficking. Mutant enzyme shows 0.18% of the wild-type activity
D227R
mutant enzyme produces only the 140000 Da enzyme form by SDS-PAGE and autoradiography compared to a 168000 Da enzyme form and a 140000 Da enzyme form of the wild-type enzyme. Mutant enzyme displays incorrect maturation and trafficking
DELTA594-945
no activity detected in cells expressing the mutant enzyme
E215A
the ratio of turnover number to KM-value for the substrate alpha-L-Glu-beta-naphthylamide is 10% of that of the wild-type enzyme. 68fold increase in KI-value for glutamate-thiol, 466fold increase in KI-value for L-glutamate phosphonic acid, 5.8fold increase in Ki-value for D-glutamate phosphonic acid
E215D
the ratio of turnover number to KM-value for the substrate alpha-L-Glu-beta-naphthylamide is 7% of that of the wild-type enzyme. 49fold increase in KI-value for glutamate-thiol, 266fold increase in KI-value for L-glutamate phosphonic acid, 2.6fold increase in Ki-value for D-glutamate phosphonic acid
E215Q
1.2fold increase in the ratio of turnover number to KM-value for the substrate alpha-L-Glu-beta-naphthylamide. 2.3fold increase in KI-value for glutamate-thiol, 5.8fold increase in KI-value for L-glutamate phosphonic acid, 8.4fold increase in Ki-value for D-glutamate phosphonic acid
H450F
mutation markedly lowers the level of enzyme activity and changes the sensitivity to Ca2+, The EC50 for Ca2+ is 0.025 mM in the wild-type enzyme and 0.279 mM in the mutant enzyme. 15.5fold increase of KI-value for glutamate-thiol in presence of 0.025 mM Ca2+, 18.9fold increase of KI-value for glutamate-thiol in presence of 4 mM Ca2+, 91.8fold increase of Ki-value for glutamate phosphonic acid in presence of 0.025 mM Ca2+, 22.1fold increase of Ki-value for glutamate phosphonic acid in presence of 4 mM Ca2+, 2.5fold increase of Ki-value for lysine-thiol in presence of 0.025 mM Ca2+, 2.76fold increase of Ki-value for lysine-thiol in presence of 4 mM Ca2+. Mutant enzyme shows a 1150fold decrease in the ratio of turnover number to Km-value for alpha-L-Glu-beta-naphthylamide in presence of 0.025 mM Ca2+. Mutant enzyme shows a 571fold decrease in the ratio of turnover number to Km-value for alpha-L-Glu-beta-naphthylamide in presence of 5 mM Ca2+
N353A
the ratio of turnover number to Km-value is 7% of the wild-type value with alpha-L-Glu-2-naphthylamide as substrate, the ratio of turnover number to Km-value is 3% of the wild-type value with angiotensin II as substrate. The Ki-value for glutamate-thiol is 9.3fold higher than the wild-type value, the Ki-value for glutamine-thiol is 4.3fold higher than the wild-type value, the Ki-value for L-glutamate phosphonic acid is 6.6fold higher than the wild-type value, the Ki-value for D-glutamate phosphonic acid is 3.4fold higher than the wild-type value, the Ki-value for captopril is 86% of the wild-type value. Mutant enzyme is similarly routed and glycosylated to the wild-type enzyme
N353D
mutant enzyme exhibits weak and unstable activity, mutant enzyme is trapped intracellularly and partially glycosylated
N353Q
the ratio of turnover number to Km-value is 4% of the wild-type value with alpha-L-Glu-2-naphthylamide as substrate, the ratio of turnover number to Km-value is 7% of the wild-type value with alpha-L-Asp-2-naphthylamide as substrate, the ratio of turnover number to Km-value is 5% of the wild-type value with angiotensin II as substrate.The Ki-value for glutamate-thiol is 6.3fold higher than the wild-type value, the Ki-value for glutamine-thiol is 4.4fold higher than the wild-type value, the Ki-value for L-glutamate phosphonic acid is 3.57fold higher than the wild-type value, the Ki-value for D-glutamate phosphonic acid is 1.5fold higher than the wild-type value, the Ki-value for captopril is 2fold higher than the wild-type value. Mutant enzyme is similarly routed and glycosylated to the wild-type enzyme
R220A
mutant enzyme produces only the 140000 Da enzyme form by SDS-PAGE and autoradiography compared to a 168000 Da enzyme form and a 140000 Da enzyme form of the wild-type enzyme. Mutant enzyme displays incorrect maturation and trafficking. Mutant enzyme shows 0.3% of the wild-type activity
R220D
mutant enzyme produces only the 140000 Da enzyme form by SDS-PAGE and autoradiography compared to a 168000 Da enzyme form and a 140000 Da enzyme form of the wild-type enzyme. Mutant enzyme displays incorrect maturation and trafficking
R220D/D227R
mutant enzyme produces only the 140000 Da enzyme form by SDS-PAGE and autoradiography compared to a 168000 Da enzyme form and a 140000 Da enzyme form of the wild-type enzyme. Mutant enzyme displays incorrect maturation and trafficking
R878A
site-directed mutagenesis, the mutant shows decreased affinity for the acidic substrate, alpha-L-glutamyl-beta-naphthylamide, with a slight decrease in substrate hydrolysis velocity either with or without Ca2+ compared to the wild-type enzyme. Analysis of the 3D models of the Arg878 mutated APAs reveals a change in the volume of the S1 subsite, which may impair the binding and/or the optimal positioning of the substrate in the active site as well as its hydrolysis
R878K
site-directed mutagenesis, the mutant shows decreased affinity for the acidic substrate, alpha-L-glutamyl-beta-naphthylamide, with a slight decrease in substrate hydrolysis velocity either with or without Ca2+ compared to the wild-type enzyme. Analysis of the 3D models of the Arg878 mutated APAs reveals a change in the volume of the S1 subsite, which may impair the binding and/or the optimal positioning of the substrate in the active site as well as its hydrolysis
T348D
the mutation leads to a modification of the hydrolysis velocity, with no change in the affinity of the recombinant enzymes for the substrate L-Glu-2-naphthylamide, either in the absence or presence of Ca2+
T348S
mutant with increased activity, the mutation leads to a modification of the hydrolysis velocity, with no change in the affinity of the recombinant enzymes for the substrate L-Glu-2-naphthylamide, either in the absence or presence of Ca2+
T348Y
mutant with strongly reduced activity, the mutation leads to a modification of the hydrolysis velocity, with no change in the affinity of the recombinant enzymes for the substrate L-Glu-2-naphthylamide, either in the absence or presence of Ca2+
additional information
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truncated form lacking cytosolic part
additional information
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construction of active site residue mutants
additional information
substitution mutations at Asp218 and Asp213 maintain the ability of APA to be activated by Ca2+ and induce a decrease of the inhibitory potencies of inhibitors homologous with acidic residues
additional information
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substitution mutations at Asp218 and Asp213 maintain the ability of APA to be activated by Ca2+ and induce a decrease of the inhibitory potencies of inhibitors homologous with acidic residues
additional information
Ca2+ activation profile of purified recombinant wild-type and mutated His-mAPAs, using an acidic substrate alpha-L-glutamyl-beta-naphthylamide, overview
additional information
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Ca2+ activation profile of purified recombinant wild-type and mutated His-mAPAs, using an acidic substrate alpha-L-glutamyl-beta-naphthylamide, overview