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show all sequences of 1.1.1.87

Evidence for an induced conformational change in the catalytic mechanism of homoisocitrate dehydrogenase for Saccharomyces cerevisiae: Characterization of the D271N mutant enzyme

Hsu, C.; West, A.H.; Cook, P.F.; Arch. Biochem. Biophys. 584, 20-27 (2015)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
Saccharomyces cerevisiae
Engineering
Amino acid exchange
Commentary
Organism
D271N
site-directed mutagenesis, mutation of a metal ion ligand and binding determinant for Mg2+, to N. The mutant enzyme shows a decrease of 520fold in V and V/Km_Mg2+, suggesting that the same step(s) limit the reaction at limiting and saturating MgHIc concentrations
Saccharomyces cerevisiae
K206M
site-directed mutagenesis, inactive mutant
Saccharomyces cerevisiae
Y150F
site-directed mutagenesis, inactive mutant
Saccharomyces cerevisiae
Inhibitors
Inhibitors
Commentary
Organism
Structure
3-acetylpyridine adenine dinucleotide 3'-phosphate
3-AcPyrADP, competitive versus NAD+
Saccharomyces cerevisiae
thiahomoisocitrate
competitive versus homoisocitrate
Saccharomyces cerevisiae
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
Michaelis-Menten kinetics, the enzyme shows a steady-state random kinetic mechanism with a preferred order of addition of Mg2+ prior to NAD+. The same step(s) limit the reaction at limiting and saturating Mg2+ concentrations. Solvent kinetic deuterium isotope effects and viscosity effects are consistent with a rate-limiting pre-catalytic conformational change at saturating reactant concentrations
Saccharomyces cerevisiae
Metals/Ions
Metals/Ions
Commentary
Organism
Structure
K+
the enzyme requires a potassium ion as an activator, for optimal binding of NAD+
Saccharomyces cerevisiae
Mg2+
required, three conserved aspartate residues, D243, D267 and D271, coordinate Mg2+, which is also coordinated to the alpha-carboxylate and alpha-hydroxyl of homoisocitrate
Saccharomyces cerevisiae
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
homoisocitrate + NAD+
Saccharomyces cerevisiae
-
2-oxoadipate + CO2 + NADH + H+
-
-
r
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Saccharomyces cerevisiae
P40495
-
-
Purification (Commentary)
Commentary
Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chrmatography
Saccharomyces cerevisiae
Reaction
Reaction
Commentary
Organism
(1R,2S)-1-hydroxybutane-1,2,4-tricarboxylate + NAD+ = 2-oxoadipate + CO2 + NADH + H+
there are 2 groups acting as acid-base catalysts in the reaction. One residue with a pKa of 6.5-7.0 serves as the general base to accept a proton as the beta-hydroxy acid is oxidized to the beta-keto acid, and this residue participates in all three of the chemical steps, acting to shuttle a proton between the C2 hydroxyl and itself. The metal ion then acts as a Lewis acid to catalyze the decarboxylation of the beta-ketoacid, with the general base donating a proton to the keto oxygen as the enol of alpha-ketoadipate is formed. A second residue with a pKa of 9.5 likely catalyzes the tautomerization step by donating a proton to the enol to give the final product. Catalytic rapid equilibrium random kinetic mechanism, overview
Saccharomyces cerevisiae
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
homoisocitrate + NAD+
-
739958
Saccharomyces cerevisiae
2-oxoadipate + CO2 + NADH + H+
-
-
-
r
additional information
the wild-type enzyme with isocitrate as the substrate is about 200times slower than with homoisocitrate
739958
Saccharomyces cerevisiae
?
-
-
-
-
Temperature Optimum [C]
Temperature Optimum [C]
Temperature Optimum Maximum [C]
Commentary
Organism
25
-
assay at
Saccharomyces cerevisiae
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
5.5
9.5
pH independence of the catalytic reaction over the range of pH 5.5-9.5
Saccharomyces cerevisiae
pH Range
pH Minimum
pH Maximum
Commentary
Organism
5.5
9.5
pH independence of the catalytic reaction over the range of pH 5.5-9.5, pH profiles
Saccharomyces cerevisiae
Cofactor
Cofactor
Commentary
Organism
Structure
NAD+
-
Saccharomyces cerevisiae
NADH
-
Saccharomyces cerevisiae
Cloned(Commentary) (protein specific)
Commentary
Organism
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
Saccharomyces cerevisiae
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
NAD+
-
Saccharomyces cerevisiae
NADH
-
Saccharomyces cerevisiae
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
D271N
site-directed mutagenesis, mutation of a metal ion ligand and binding determinant for Mg2+, to N. The mutant enzyme shows a decrease of 520fold in V and V/Km_Mg2+, suggesting that the same step(s) limit the reaction at limiting and saturating MgHIc concentrations
Saccharomyces cerevisiae
K206M
site-directed mutagenesis, inactive mutant
Saccharomyces cerevisiae
Y150F
site-directed mutagenesis, inactive mutant
Saccharomyces cerevisiae
Inhibitors (protein specific)
Inhibitors
Commentary
Organism
Structure
3-acetylpyridine adenine dinucleotide 3'-phosphate
3-AcPyrADP, competitive versus NAD+
Saccharomyces cerevisiae
thiahomoisocitrate
competitive versus homoisocitrate
Saccharomyces cerevisiae
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
Michaelis-Menten kinetics, the enzyme shows a steady-state random kinetic mechanism with a preferred order of addition of Mg2+ prior to NAD+. The same step(s) limit the reaction at limiting and saturating Mg2+ concentrations. Solvent kinetic deuterium isotope effects and viscosity effects are consistent with a rate-limiting pre-catalytic conformational change at saturating reactant concentrations
Saccharomyces cerevisiae
Metals/Ions (protein specific)
Metals/Ions
Commentary
Organism
Structure
K+
the enzyme requires a potassium ion as an activator, for optimal binding of NAD+
Saccharomyces cerevisiae
Mg2+
required, three conserved aspartate residues, D243, D267 and D271, coordinate Mg2+, which is also coordinated to the alpha-carboxylate and alpha-hydroxyl of homoisocitrate
Saccharomyces cerevisiae
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
homoisocitrate + NAD+
Saccharomyces cerevisiae
-
2-oxoadipate + CO2 + NADH + H+
-
-
r
Purification (Commentary) (protein specific)
Commentary
Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chrmatography
Saccharomyces cerevisiae
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
homoisocitrate + NAD+
-
739958
Saccharomyces cerevisiae
2-oxoadipate + CO2 + NADH + H+
-
-
-
r
additional information
the wild-type enzyme with isocitrate as the substrate is about 200times slower than with homoisocitrate
739958
Saccharomyces cerevisiae
?
-
-
-
-
Temperature Optimum [C] (protein specific)
Temperature Optimum [C]
Temperature Optimum Maximum [C]
Commentary
Organism
25
-
assay at
Saccharomyces cerevisiae
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
5.5
9.5
pH independence of the catalytic reaction over the range of pH 5.5-9.5
Saccharomyces cerevisiae
pH Range (protein specific)
pH Minimum
pH Maximum
Commentary
Organism
5.5
9.5
pH independence of the catalytic reaction over the range of pH 5.5-9.5, pH profiles
Saccharomyces cerevisiae
General Information
General Information
Commentary
Organism
evolution
the enzyme is a member of the family of pyridine dinucleotide-dependent beta-hydroxyacid oxidative decarboxylating dehydrogenases, specifically the family that has (R)-beta-hydroxyacid substrates, including isocitrate dehydrogenase (ICDH) among others. Superposition of available structures of the malic enzyme, isopropylmalate dehydrogenase, IcDH, and HIcDH show a similar overall geometry of residues in the substrate and metal ion binding sites. A Lys (general base)-Tyr (general acid) pair is conserved among these enzymes. The similar structural geometry in the active site suggests a similar general chemical mechanism. Three aspartate residues are conserved in the active sites of all HIcDHs sequenced to data, and are also conserved across the family of pyridine nucleotide-dependent oxidative decarboxylases including malic enzyme
Saccharomyces cerevisiae
metabolism
homoisocitrate dehydrogenase catalyzes the fourth step of the alpha-aminadipate pathway, the NAD+-dependent conversion of homoisocitrate to alpha-ketoadipate
Saccharomyces cerevisiae
additional information
a conformational change to close the active site and organize the active site for catalysis contributes to rate limitation of the overall reaction of the Saccharomyces cerevisiae enzyme HIcDH. Residues K206 and Y150 of ScHIcDH are a Lys-Tyr pair in the active site acting as the general base and general acid in the reaction. A slow conformational change is required to close the site upon the binding of MgHIc prior to catalysis. With the slow substrate isocitrate, hydride transfer and decarboxylation steps contribute to rate limitation, and the decarboxylation step is the slower of the two
Saccharomyces cerevisiae
General Information (protein specific)
General Information
Commentary
Organism
evolution
the enzyme is a member of the family of pyridine dinucleotide-dependent beta-hydroxyacid oxidative decarboxylating dehydrogenases, specifically the family that has (R)-beta-hydroxyacid substrates, including isocitrate dehydrogenase (ICDH) among others. Superposition of available structures of the malic enzyme, isopropylmalate dehydrogenase, IcDH, and HIcDH show a similar overall geometry of residues in the substrate and metal ion binding sites. A Lys (general base)-Tyr (general acid) pair is conserved among these enzymes. The similar structural geometry in the active site suggests a similar general chemical mechanism. Three aspartate residues are conserved in the active sites of all HIcDHs sequenced to data, and are also conserved across the family of pyridine nucleotide-dependent oxidative decarboxylases including malic enzyme
Saccharomyces cerevisiae
metabolism
homoisocitrate dehydrogenase catalyzes the fourth step of the alpha-aminadipate pathway, the NAD+-dependent conversion of homoisocitrate to alpha-ketoadipate
Saccharomyces cerevisiae
additional information
a conformational change to close the active site and organize the active site for catalysis contributes to rate limitation of the overall reaction of the Saccharomyces cerevisiae enzyme HIcDH. Residues K206 and Y150 of ScHIcDH are a Lys-Tyr pair in the active site acting as the general base and general acid in the reaction. A slow conformational change is required to close the site upon the binding of MgHIc prior to catalysis. With the slow substrate isocitrate, hydride transfer and decarboxylation steps contribute to rate limitation, and the decarboxylation step is the slower of the two
Saccharomyces cerevisiae
Other publictions for EC 1.1.1.87
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [C]
Temperature Range [C]
Temperature Stability [C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [C] (protein specific)
Temperature Range [C] (protein specific)
Temperature Stability [C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
746605
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4
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1
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1
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685539
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Saccharomyces cerevisiae
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1
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687988
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Mus musculus
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508
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667934
Yamamoto
Substrate specificity analysis ...
Deinococcus radiodurans, Saccharomyces cerevisiae
Bioorg. Med. Chem.
15
1346-1355
2007
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1
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11
20
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2
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7
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20
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9
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24
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2
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686950
Sandell
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Mus musculus
Genes Dev.
21
1113-1124
2007
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660907
Miyazaki
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Pyrococcus horikoshii
Biochem. Biophys. Res. Commun.
331
341-346
2005
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660918
Miyazaki
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Deinococcus radiodurans
Biochem. Biophys. Res. Commun.
336
596-602
2005
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4
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7
1
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669109
Miyazaki
Crystal structure of tetrameri ...
Thermus thermophilus
J. Bacteriol.
187
6779-6788
2005
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1
1
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2
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1
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2
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1
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1
1
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1
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1
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1
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656127
Miyazaki
Characterization of homoisocit ...
Thermus thermophilus, Thermus thermophilus HB27 / ATCC BAA-163 / DSM 7039
J. Biol. Chem.
278
1864-1871
2003
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1
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1
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27
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8
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8
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644538
Garrad
Lysine biosynthesis in selecte ...
Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, Saccharomyces cerevisiae
J. Bacteriol.
174
7379-7384
1992
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3
1
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4
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