Cloned (Comment) | Organism |
---|---|
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Saccharomyces cerevisiae |
Protein Variants | Comment | 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 | Comment | 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 Maximum [mM] | Substrate | Comment | 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 | Comment | 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 | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
homoisocitrate + NAD+ | Saccharomyces cerevisiae | - |
2-oxoadipate + CO2 + NADH + H+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Saccharomyces cerevisiae | P40495 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chrmatography | Saccharomyces cerevisiae |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
(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 | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
homoisocitrate + NAD+ | - |
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 | Saccharomyces cerevisiae | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
3-carboxy-2-hydroxyadipate dehydrogenase | - |
Saccharomyces cerevisiae |
HICDH | - |
Saccharomyces cerevisiae |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Saccharomyces cerevisiae |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
5.5 | 9.5 | pH independence of the catalytic reaction over the range of pH 5.5-9.5 | Saccharomyces cerevisiae |
pH Minimum | pH Maximum | Comment | 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 | Comment | Organism | Structure |
---|---|---|---|
NAD+ | - |
Saccharomyces cerevisiae | |
NADH | - |
Saccharomyces cerevisiae |
General Information | Comment | 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 |