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Literature summary extracted from

  • Zeczycki, T.N.; Menefee, A.L.; Jitrapakdee, S.; Wallace, J.C.; Attwood, P.V.; St Maurice, M.; Cleland, W.W.
    Activation and inhibition of pyruvate carboxylase from Rhizobium etli (2011), Biochemistry, 50, 9694-9707.
    View publication on PubMedView publication on EuropePMC

Activating Compound

EC Number Activating Compound Comment Organism Structure
6.4.1.1 acetyl-CoA nonessential activator. Both acetyl-CoA and Mg2+ assist in coupling the MgATP-dependent carboxylation of biotin in the biotin carboxylase (BC) domain with pyruvate carboxylation in the carboxyl transferase (CT) domain. Absence of acetyl-CoA results in only 9% of fully activated enzyme. Acetyl-CoA also has a noticeable effect on the activity of the oxamate-induced decarboxylation of oxaloacetate but no effect on the rate of MgADP phosphorylation by carbamoyl phosphate Rhizobium etli

Cloned(Commentary)

EC Number Cloned (Comment) Organism
6.4.1.1 expressed in Escherichia coli as a His-tagged fusion protein Rhizobium etli

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
6.4.1.1 crystal structures of mutant T882A pyruvate carboxylase are determined cocrystallized with phosphonoacetate and MgADP Rhizobium etli

Protein Variants

EC Number Protein Variants Comment Organism
6.4.1.1 K1119Q mutant that lacks tethered biotin. Addition of 10 mM biotin increases the kcat of MgATP hydrolysis to rates observed for wild-type RePC in the absence of free biotin. This rate increase, coupled with a 35fold decrease in the Km for MgATP, results in a nearly 1000fold increase in the catalytic efficiency of the mutant K1119Q RePC catalyzed reaction when 10 mM free biotin is added Rhizobium etli
6.4.1.1 T882A T882 mutant is a tetrameric holoenzyme where positioning of the tethered biotin favors placement in the BC domain. Free biotin increases the kcat for both the wild-type and the T882A mutant RePC-catalyzed reactions without having a major effect on the Km for MgATP. Crystal structures of mutant T882A pyruvate carboxylase are determined cocrystallized with phosphonoacetate and MgADP Rhizobium etli

Inhibitors

EC Number Inhibitors Comment Organism Structure
6.4.1.1 Phosphonoacetate
-
 Rhizobium etli

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.4.1.1 additional information
-
 additional information 0.5 mM Mg2+: Vmax (micromol/mg/min) 1.16, Ka (acetyl-CoA) 0.038 mM/1 mM Mg2+: Vmax (micromol/mg/min) 1.39, Ka (acetyl-CoA) 0.026 mM/1.5 mM Mg2+: Vmax (micromol/mg/min) 2.59, Ka (acetyl-CoA) 0.025 mM/3 mM Mg2+: Vmax (micromol/mg/min) 5.7, Ka (acetyl-CoA) 0.01 mM Rhizobium etli
6.4.1.1 0.018
-
 ATP pH 7.5, 25°C, wild-type, 10 mM biotin, pyruvate carboxylation Rhizobium etli
6.4.1.1 0.022
-
 ATP pH 7.5, 25°C, wild-type, no biotin, pyruvate carboxylation Rhizobium etli
6.4.1.1 0.04
-
 ATP pH 7.5, 25°C, mutant K119Q, 10 mM biotin, pyruvate carboxylation Rhizobium etli
6.4.1.1 0.054
-
 ATP pH 7.5, 25°C, mutant T882A, no biotin, pyruvate carboxylation Rhizobium etli
6.4.1.1 0.09
-
 ATP pH 7.5, 25°C, mutant T882A, 10 mM biotin, pyruvate carboxylation Rhizobium etli
6.4.1.1 0.1 2 ADP pH 7.5, 25°C, wild-type, reverse reaction Rhizobium etli
6.4.1.1 0.145
-
 ATP pH 7.5, 25°C, wild-type, pyruvate carboxylation Rhizobium etli
6.4.1.1 0.15
-
 pyruvate 5 mM Mg2+, pH 7.5, 25°C, wild-type, pyruvate carboxylation, Vmax: 3.98 micromol/mg/min Rhizobium etli
6.4.1.1 0.15
-
 pyruvate pH 7.5, 25°C, wild-type, pyruvate carboxylation Rhizobium etli
6.4.1.1 1.2
-
 ATP pH 7.5, 25°C, mutant K119Q, no biotin, pyruvate carboxylation Rhizobium etli
6.4.1.1 2.65
-
 pyruvate 3 mM Mg2+, pH 7.5, 25°C, wild-type, pyruvate carboxylation, Vmax: 3.18 micromol/mg/min Rhizobium etli
6.4.1.1 3
-
 pyruvate 1.5 mM Mg2+, pH 7.5, 25°C, wild-type, pyruvate carboxylation, Vmax: 1.41 micromol/mg/min Rhizobium etli
6.4.1.1 3.1
-
 pyruvate 0.7 mM Mg2+, pH 7.5, 25°C, wild-type, pyruvate carboxylation, Vmax: 0.69 micromol/mg/min Rhizobium etli
6.4.1.1 10.8
-
 HCO3- pH 7.5, 25°C, wild-type, pyruvate carboxylation Rhizobium etli

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.4.1.1 Mg2+ essential activator. Both acetyl-CoA and Mg2+ assist in coupling the MgATP-dependent carboxylation of biotin in the biotin carboxylase (BC) domain with pyruvate carboxylation in the carboxyl transferase (CT) domain. High Mg2+ concentration (above 7 mM) inhibits pyruvate carboxylation and MgATP cleavage but no inhibition of MgADP phosphorylation reaction Rhizobium etli

Organism

EC Number Organism UniProt Comment Textmining
6.4.1.1 Rhizobium etli Q2K340
-
-
6.4.1.1 Rhizobium etli CFN 42 Q2K340
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
6.4.1.1 using Ni-NTA chromatography Rhizobium etli

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.4.1.1 ATP + pyruvate + HCO3-
-
 Rhizobium etli ADP + phosphate + oxaloacetate
-
 r
6.4.1.1 ATP + pyruvate + HCO3-
-
 Rhizobium etli CFN 42 ADP + phosphate + oxaloacetate
-
 r

Synonyms

EC Number Synonyms Comment Organism
6.4.1.1 pyruvate carboxylase
-
 Rhizobium etli
6.4.1.1 RePC
-
 Rhizobium etli

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
6.4.1.1 25
-
 assay at Rhizobium etli

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
6.4.1.1 0.0013
-
 ATP pH 7.5, 25°C, mutant K119Q, no biotin, pyruvate carboxylation Rhizobium etli
6.4.1.1 0.013
-
 ADP pH 7.5, 25°C, wild-type, reverse reaction Rhizobium etli
6.4.1.1 0.04
-
 ATP pH 7.5, 25°C, mutant K119Q, 10 mM biotin, pyruvate carboxylation Rhizobium etli
6.4.1.1 0.043
-
 ATP pH 7.5, 25°C, wild-type, no biotin, pyruvate carboxylation Rhizobium etli
6.4.1.1 0.05
-
 ATP pH 7.5, 25°C, wild-type, 10 mM biotin, pyruvate carboxylation Rhizobium etli
6.4.1.1 0.23
-
 ATP pH 7.5, 25°C, mutant T882A, no biotin, pyruvate carboxylation Rhizobium etli
6.4.1.1 0.6
-
 ATP pH 7.5, 25°C, mutant T882A, 10 mM biotin, pyruvate carboxylation Rhizobium etli
6.4.1.1 6.6
-
 ATP pH 7.5, 25°C, wild-type, pyruvate carboxylation Rhizobium etli
6.4.1.1 7.33
-
 pyruvate pH 7.5, 25°C, wild-type, pyruvate carboxylation Rhizobium etli
6.4.1.1 11.6
-
 HCO3- pH 7.5, 25°C, wild-type, pyruvate carboxylation Rhizobium etli

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
6.4.1.1 7.5
-
 assay at Rhizobium etli

Cofactor

EC Number Cofactor Comment Organism Structure
6.4.1.1 biotin
-
 Rhizobium etli

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
6.4.1.1 0.0011
-
 ATP pH 7.5, 25°C, mutant K119Q, no biotin, pyruvate carboxylation Rhizobium etli
6.4.1.1 0.11
-
 ADP pH 7.5, 25°C, wild-type, reverse reaction Rhizobium etli
6.4.1.1 1.08
-
 HCO3- pH 7.5, 25°C, wild-type, pyruvate carboxylation Rhizobium etli
6.4.1.1 1.125
-
 ATP pH 7.5, 25°C, mutant K119Q, 10 mM biotin, pyruvate carboxylation Rhizobium etli
6.4.1.1 1.91
-
 ATP pH 7.5, 25°C, wild-type, no biotin, pyruvate carboxylation Rhizobium etli
6.4.1.1 2.78
-
 ATP pH 7.5, 25°C, wild-type, 10 mM biotin, pyruvate carboxylation Rhizobium etli
6.4.1.1 4.33
-
 ATP pH 7.5, 25°C, mutant T882A, no biotin, pyruvate carboxylation Rhizobium etli
6.4.1.1 7.16
-
 ATP pH 7.5, 25°C, mutant T882A, 10 mM biotin, pyruvate carboxylation Rhizobium etli
6.4.1.1 44.5
-
 ATP pH 7.5, 25°C, wild-type, pyruvate carboxylation Rhizobium etli
6.4.1.1 48.3
-
 pyruvate pH 7.5, 25°C, wild-type, pyruvate carboxylation Rhizobium etli