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Literature summary extracted from

  • Zhang, P.; Ma, J.; Zhang, Z.; Zha, M.; Xu, H.; Zhao, G.; Ding, J.
    Molecular basis of the inhibitor selectivity and insights into the feedback inhibition mechanism of citramalate synthase from Leptospira interrogans (2009), Biochem. J., 421, 133-143.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.3.3.21
-
 Leptospira interrogans
2.3.3.21 full-length and mutant CMS Leptospira interrogans

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.3.3.21 C-terminal regulatory domain of CMS (CMSC) in complex with isoleucine, by hanging-drop vapour diffusion method. Crystals of CMSC belong to space group C222 (type I) with unit cell parameters a = 62.6 A, b = 98.4 A and c = 40.1 A, refined to 2.5 A resolution. Type II crystals of CMSC grown at room temperature belong to space group C2 with unit cell parameters a = 61.3 A, b = 97.9 A, c = 40.0 A and beta = 91.4, refined to 2.0 A resolution. Type III crystals of CMSC grown at 4°C belong to space group C222 with unit cell parameters a = 108.2 A, b = 118.6 A and c = 63.6 A, refined to 2.7 A resolution. CMSC consists of six beta-strands forming two anti-parallel beta-sheets and two alpha-helices and assumes a betaalphabeta three-layer sandwich structure. The inhibitor isoleucine is bound in a pocket at the dimer interface and has both hydrophobic and hydrogen-bonding interactions with several conserved residues of both subunits Leptospira interrogans
2.3.3.21 hanging-drop vapor diffusion, in 20 mM Tris-HCl, pH 8.4 with 50 mM NaCl Leptospira interrogans

Protein Variants

EC Number Protein Variants Comment Organism
2.3.3.21 D431A changes hydrophobic residues at inhibitor binding site, minor effect on Km for acetyl-CoA but moderate decrease of kcat in the absence of inhibitor isoleucine, severely decreased inhibition by isoleucine (increase of IC50 and Ki values) Leptospira interrogans
2.3.3.21 D431A has markedly increased IC50 and Ki values for isoleucine Leptospira interrogans
2.3.3.21 H400A/H408A prevents Zn2+-binding and enables dimer instead of tetramer formation, no change in enzymatic activity or the inhibition by isoleucine Leptospira interrogans
2.3.3.21 H400A/H408A mutant exists purely as a dimer in solution. Is inhibited by isoleucine with comparable IC50 and Ki values as the wild-type enzyme, the double mutations have very little effects on the binding (Km) of acetyl-CoA and the kcat. Binding of isoleucine with the mutant does not affect the dimeric state of the enzyme Leptospira interrogans
2.3.3.21 I458A changes hydrophobic residues at inhibitor binding site, minor effect on Km for acetyl-CoA but moderate decrease of kcat in the absence of inhibitor isoleucine, no inhibition by isoleucine, no binding of isoleucine Leptospira interrogans
2.3.3.21 I458A inhibition by isoleucine is severely decreased. Lower Kcat/Km as the wild-type Leptospira interrogans
2.3.3.21 L451V changes hydrophobic residues at inhibitor binding site, minor effect on Km for acetyl-CoA but moderate decrease of kcat in the absence of inhibitor isoleucine, severely decreased inhibition by isoleucine (increase of IC50 and Ki values) Leptospira interrogans
2.3.3.21 L451V inhibition by isoleucine is severely decreased Leptospira interrogans
2.3.3.21 P493A changes hydrophobic residues at inhibitor binding site, minor effect on Km for acetyl-CoA but moderate decrease of kcat in the absence of inhibitor isoleucine, severely decreased inhibition by isoleucine (increase of IC50 and Ki values) Leptospira interrogans
2.3.3.21 P493A inhibition by isoleucine is greatly decreased Leptospira interrogans
2.3.3.21 Q495A changes hydrophobic residues at inhibitor binding site, minor effect on Km for acetyl-CoA but moderate decrease of kcat in the absence of inhibitor isoleucine, severely decreased inhibition by isoleucine (increase of IC50 and Ki values) Leptospira interrogans
2.3.3.21 Q495A exhibits significantly increased IC50 and Ki values for isoleucine Leptospira interrogans
2.3.3.21 T464A changes hydrophobic residues at inhibitor binding site, minor effect on Km for acetyl-CoA but moderate decrease of kcat in the absence of inhibitor isoleucine, minor effect on IC50 and Ki values for isoleucine Leptospira interrogans
2.3.3.21 T464A minor effect on the IC50 and Ki values for isoleucine Leptospira interrogans
2.3.3.21 V468A changes hydrophobic residues at inhibitor binding site, minor effect on Km for acetyl-CoA but moderate decrease of kcat in the absence of inhibitor isoleucine, slightly increased inhibition by isoleucine, and inhibition by leucine Leptospira interrogans
2.3.3.21 V468A inhibition by isoleucine is slightly decreased Leptospira interrogans
2.3.3.21 Y430L changes hydrophobic residues at inhibitor binding site, minor effect on Km for acetyl-CoA but moderate decrease of kcat in the absence of inhibitor isoleucine, severely decreased inhibition by isoleucine (increase of IC50 and Ki values) Leptospira interrogans
2.3.3.21 Y430L inhibition by isoleucine is severely decreased Leptospira interrogans
2.3.3.21 Y454A changes hydrophobic residues at inhibitor binding site, minor effect on Km for acetyl-CoA but moderate decrease of kcat in the absence of inhibitor isoleucine, severely decreased inhibition by isoleucine (increase of IC50 and Ki values), slight inhibition by leucine Leptospira interrogans
2.3.3.21 Y454A inhibition by isoleucine is severely decreased. Lower Kcat/Km as the wild-type Leptospira interrogans

Inhibitors

EC Number Inhibitors Comment Organism Structure
2.3.3.21 isoleucine end product feedback inhibition, competitive inhibition of the binding of pyruvate and acetyl-CoA; inhibits in a concentration-dependent manner. High selectivity of CMS for isoleucine over leucine is primarily dictated by the residues, Tyr430, Leu451, Tyr454, Ile458 and Val468, that form a hydrophobic pocket to accommodate the side chain of the inhibitor. Binding of isoleucine affects the binding of the substrate and coenzyme at the active site, possibly via conformational change of the dimer interface of the regulatory domain, leading to inhibition of the catalytic reaction, in a typical pattern of K-type inhibition Leptospira interrogans
2.3.3.21 leucine inhibits only the mutants Y454A and V468A Leptospira interrogans
2.3.3.21 additional information mutant I458A is not inhibited by isoleucine, leucine does not inhibit the wild-type and the mutants H400A/H408A, Y430L, L451V, I458A, D431A, T464A, P493A, and Q495A Leptospira interrogans

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.3.3.21 0.06
-
 pyruvate wild-type, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 0.06
-
 pyruvate wild-type, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 0.094
-
 pyruvate wild-type, 1 microM isoleucine, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 0.094
-
 pyruvate wild-type, in the presence of 0.001 mM isoleucine, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 0.178
-
 pyruvate wild-type, 4 microM isoleucine, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 0.178
-
 pyruvate wild-type, in the presence of 0.004 mM isoleucine, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 0.626
-
 acetyl-CoA mutant T464A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 0.626
-
 acetyl-CoA T464A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 0.629
-
 acetyl-CoA D431A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 0.629
-
 acetyl-CoA mutant D431A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 0.712
-
 acetyl-CoA mutant Q495A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 0.712
-
 acetyl-CoA Q495A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 0.743
-
 acetyl-CoA mutant P493A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 0.743
-
 acetyl-CoA P493A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 0.803
-
 acetyl-CoA mutant Y454A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 0.803
-
 acetyl-CoA Y454A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 0.838
-
 acetyl-CoA I458A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 0.838
-
 acetyl-CoA mutant I458A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 1.097
-
 acetyl-CoA mutant Y430L, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 1.097
-
 acetyl-CoA Y430L 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 1.116
-
 acetyl-CoA mutant V468A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 1.116
-
 acetyl-CoA V468A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 1.118
-
 acetyl-CoA wild-type, 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 1.118
-
 acetyl-CoA wild-type, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 1.554
-
 acetyl-CoA H400A/H408A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 1.554
-
 acetyl-CoA mutant H400A/H408A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 1.711
-
 acetyl-CoA L451V 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 1.711
-
 acetyl-CoA mutant L451V, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 2.272
-
 acetyl-CoA wild-type, 20 microM isoleucine, 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 2.272
-
 acetyl-CoA wild-type, in the presence of 0.02 mM isoleucine, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 4.246
-
 acetyl-CoA wild-type, 60 microM isoleucine, 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 4.246
-
 acetyl-CoA wild-type, in the presence of 0.06 mM isoleucine, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.3.3.21 Mn2+
-
 Leptospira interrogans
2.3.3.21 Zn2+ bound, responsible for tetramer formation, removal or mutation of the binding sites by replacement of the histidine 400 and 408 by alanine leads to dimer formation Leptospira interrogans
2.3.3.21 Zn2+ type II crystal structure contains two Zn2+ ions (which are located on the 2fold symmetry axis with an occupancy of 0.5) in the asymmetric unit. The type III crystal structure contains one Zn2+ ion in the asymmetric unit Leptospira interrogans

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.3.3.21 120000
-
 wild-type full-length, Zn2+ removed by EDTA, gel filtration analysis Leptospira interrogans
2.3.3.21 128000
-
 H400A/H408A mutant enzyme (preventing Zn2+-binding), gel filtration analysis Leptospira interrogans
2.3.3.21 310000
-
 wild-type full-length, gel filtration analysis Leptospira interrogans

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.3.3.21 acetyl-CoA + pyruvate + H2O Leptospira interrogans
-
 CoA + (2R)-2-hydroxy-2-methylbutanedioate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.3.3.21 Leptospira interrogans
-
-
-
2.3.3.21 Leptospira interrogans
-
 leptospirosis pathogen
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.3.3.21
-
 Leptospira interrogans
2.3.3.21 full-length and mutant CMS Leptospira interrogans

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.3.3.21 acetyl-CoA + pyruvate + H2O
-
 Leptospira interrogans CoA + (2R)-2-hydroxy-2-methylbutanedioate
-
 ?
2.3.3.21 acetyl-CoA + pyruvate + H2O
-
 Leptospira interrogans CoA + (R)-citramalate
-
 ?

Subunits

EC Number Subunits Comment Organism
2.3.3.21 dimer 2 * 120000, a tetramer of 2 head to head dimers of the smaller subunit LiCMSC are formed in the presence of Zn2+ Leptospira interrogans
2.3.3.21 dimer H400A/H408A mutant in solution, gel filtration Leptospira interrogans
2.3.3.21 tetramer purified wild-type in solution, gel filtration. Is presumably a tetramer due to the presence of Zn2+ co-purified with the enzyme from the expression system Leptospira interrogans

Synonyms

EC Number Synonyms Comment Organism
2.3.3.21 citramalate synthase
-
 Leptospira interrogans
2.3.3.21 CMS
-
 Leptospira interrogans
2.3.3.21 LiCMS
-
 Leptospira interrogans
2.3.3.21 LiCMSC regulatory domain of C-terminus of LiCMS Leptospira interrogans

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.3.3.21 0.6
-
 acetyl-CoA mutant Y454A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 0.6
-
 acetyl-CoA Y454A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 0.7
-
 acetyl-CoA I458A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 0.7
-
 acetyl-CoA mutant I458A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 2
-
 acetyl-CoA D431A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 2
-
 acetyl-CoA mutant D431A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 2.4
-
 acetyl-CoA L451V 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 2.4
-
 acetyl-CoA mutant L451V, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 2.4
-
 acetyl-CoA mutant T464A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 2.4
-
 acetyl-CoA T464A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 3.7
-
 acetyl-CoA mutant Q495A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 3.7
-
 acetyl-CoA Q495A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 4
-
 acetyl-CoA mutant P493A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 4
-
 acetyl-CoA P493A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 4.5
-
 acetyl-CoA mutant Y430L, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 4.5
-
 acetyl-CoA Y430L 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 5
-
 acetyl-CoA mutant V468A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 5
-
 acetyl-CoA V468A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 9.1
-
 acetyl-CoA H400A/H408A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 9.1
-
 acetyl-CoA mutant H400A/H408A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 9.1
-
 pyruvate wild-type, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 9.1
-
 pyruvate wild-type, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 9.2
-
 pyruvate wild-type, 1 microM isoleucine, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 9.2
-
 pyruvate wild-type, in the presence of 0.001 mM isoleucine, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 9.3
-
 pyruvate wild-type, 4 microM isoleucine, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 9.3
-
 pyruvate wild-type, in the presence of 0.004 mM isoleucine, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 10.3
-
 acetyl-CoA wild-type, 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 10.3
-
 acetyl-CoA wild-type, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 10.6
-
 acetyl-CoA wild-type, 20 microM isoleucine, 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 10.6
-
 acetyl-CoA wild-type, in the presence of 0.02 mM isoleucine 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 11.1
-
 acetyl-CoA wild-type, 60 microM isoleucine, 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 11.1
-
 acetyl-CoA wild-type, in the presence of 0.06 mM isoleucine, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
2.3.3.21 0.009
-
 isoleucine mutant V468A, calculated from the Cheng-Prusoff equation Leptospira interrogans
2.3.3.21 0.009
-
 isoleucine V468A 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 0.019
-
 isoleucine wild-type, 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 0.019
-
 isoleucine wild-type, calculated from the Cheng-Prusoff equation Leptospira interrogans
2.3.3.21 0.024
-
 isoleucine mutant T464A, calculated from the Cheng-Prusoff equation Leptospira interrogans
2.3.3.21 0.024
-
 isoleucine T464A 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 0.043
-
 isoleucine H400A/H408A 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 0.043
-
 isoleucine mutant H400A/H408A, calculated from the Cheng-Prusoff equation Leptospira interrogans
2.3.3.21 0.075
-
 leucine mutant V468A, 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 0.075
-
 leucine mutant V468A, calculated from the Cheng-Prusoff equation Leptospira interrogans
2.3.3.21 0.252
-
 isoleucine L451V 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 0.252
-
 isoleucine mutant L451V, calculated from the Cheng-Prusoff equation Leptospira interrogans
2.3.3.21 0.382
-
 isoleucine D431A 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 0.382
-
 isoleucine mutant D431A, calculated from the Cheng-Prusoff equation Leptospira interrogans
2.3.3.21 0.421
-
 isoleucine mutant Y454A, calculated from the Cheng-Prusoff equation Leptospira interrogans
2.3.3.21 0.421
-
 isoleucine Y454A 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 0.581
-
 isoleucine mutant P493A, calculated from the Cheng-Prusoff equation Leptospira interrogans
2.3.3.21 0.581
-
 isoleucine P493A 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 0.715
-
 isoleucine mutant Q495A, calculated from the Cheng-Prusoff equation Leptospira interrogans
2.3.3.21 0.715
-
 isoleucine Q495A 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 1.092
-
 isoleucine mutant Y430L, calculated from the Cheng-Prusoff equation Leptospira interrogans
2.3.3.21 1.092
-
 isoleucine Y430L 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 1.302
-
 leucine mutant Y454A, 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 1.302
-
 leucine mutant Y454A, calculated from the Cheng-Prusoff equation Leptospira interrogans

IC50 Value

EC Number IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
2.3.3.21 0.041
-
 mutant V468A, in the presence of 0.6 mM pyruvate, 4 mM acetyl-CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS and a varied concentration of isoleucine (approx. 0-10 mM) in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans isoleucine
2.3.3.21 0.041
-
 V468A 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans isoleucine
2.3.3.21 0.086
-
 wild-type, 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans isoleucine
2.3.3.21 0.086
-
 wild-type, in the presence of 0.6 mM pyruvate, 4 mM acetyl-CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS and a varied concentration of isoleucine (approx. 0-10 mM) in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans isoleucine
2.3.3.21 0.153
-
 H400A/H408A 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans isoleucine
2.3.3.21 0.153
-
 mutant H400A/H408A, in the presence of 0.6 mM pyruvate, 4 mM acetyl-CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS and a varied concentration of isoleucine (approx. 0-10 mM) in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans isoleucine
2.3.3.21 0.177
-
 mutant T464A, in the presence of 0.6 mM pyruvate, 4 mM acetyl-CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS and a varied concentration of isoleucine (approx. 0-10 mM) in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans isoleucine
2.3.3.21 0.177
-
 T464A 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans isoleucine
2.3.3.21 0.343
-
 mutant V468A, 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans leucine
2.3.3.21 0.343
-
 mutant V468A, in the presence of 0.6 mM pyruvate, 4 mM acetyl-CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS and a varied concentration of leucine (approx. 0-20 mM) in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans leucine
2.3.3.21 0.839
-
 L451V 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans isoleucine
2.3.3.21 0.839
-
 mutant L451V, in the presence of 0.6 mM pyruvate, 4 mM acetyl-CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS and a varied concentration of isoleucine (approx. 0-10 mM) in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans isoleucine
2.3.3.21 2.519
-
 mutant Y454A, in the presence of 0.6 mM pyruvate, 4 mM acetyl-CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS and a varied concentration of isoleucine (approx. 0-10 mM) in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans isoleucine
2.3.3.21 2.519
-
 Y454A 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans isoleucine
2.3.3.21 2.815
-
 D431A 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans isoleucine
2.3.3.21 2.815
-
 mutant D431A, in the presence of 0.6 mM pyruvate, 4 mM acetyl-CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS and a varied concentration of isoleucine (approx. 0-10 mM) in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans isoleucine
2.3.3.21 3.704
-
 mutant P493A, in the presence of 0.6 mM pyruvate, 4 mM acetyl-CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS and a varied concentration of isoleucine (approx. 0-10 mM) in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans isoleucine
2.3.3.21 3.704
-
 P493A 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans isoleucine
2.3.3.21 4.733
-
 mutant Q495A, in the presence of 0.6 mM pyruvate, 4 mM acetyl-CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS and a varied concentration of isoleucine (approx. 0-10 mM) in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans isoleucine
2.3.3.21 4.733
-
 Q495A 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans isoleucine
2.3.3.21 5.073
-
 mutant Y430L, in the presence of 0.6 mM pyruvate, 4 mM acetyl-CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS and a varied concentration of isoleucine (approx. 0-10 mM) in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans isoleucine
2.3.3.21 5.073
-
 Y430L 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans isoleucine
2.3.3.21 7.786
-
 mutant Y454A, 0.6 mM pyruvate, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, variable inhibitor concentrations, 0.1 M HEPES, pH 7.7 Leptospira interrogans leucine
2.3.3.21 7.786
-
 mutant Y454A, in the presence of 0.6 mM pyruvate, 4 mM acetyl-CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS and a varied concentration of leucine (approx. 0-20 mM) in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans leucine

General Information

EC Number General Information Comment Organism
2.3.3.21 metabolism first step in isoleucine biosynthesis Leptospira interrogans

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.3.3.21 0.74
-
 acetyl-CoA mutant Y454A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 0.8
-
 acetyl-CoA mutant I458A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 1.4
-
 acetyl-CoA L451V 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 1.4
-
 acetyl-CoA mutant L451V, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 1.5
-
 pyruvate wild-type, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 2.6
-
 acetyl-CoA wild-type, 60 microM isoleucine, 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 2.6
-
 acetyl-CoA wild-type, in the presence of 0.06 mM isoleucine, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 3.2
-
 acetyl-CoA D431A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 3.2
-
 acetyl-CoA mutant D431A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 3.8
-
 acetyl-CoA mutant T464A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 3.8
-
 acetyl-CoA T464A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 4.1
-
 acetyl-CoA mutant Y430L, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 4.1
-
 acetyl-CoA Y430L 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 4.5
-
 acetyl-CoA mutant V468A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 4.5
-
 acetyl-CoA V468A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 4.6
-
 acetyl-CoA wild-type, 20 microM isoleucine, 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 4.6
-
 acetyl-CoA wild-type, in the presence of 0.02 mM isoleucine, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 5.2
-
 acetyl-CoA mutant Q495A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 5.2
-
 acetyl-CoA Q495A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 5.2
-
 pyruvate wild-type, 4 microM isoleucine, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 5.4
-
 acetyl-CoA mutant P493A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 5.4
-
 acetyl-CoA P493A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 5.9
-
 acetyl-CoA H400A/H408A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 5.9
-
 acetyl-CoA mutant H400A/H408A, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 7.4
-
 acetyl-CoA Y454A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 8
-
 acetyl-CoA I458A 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 9.2
-
 acetyl-CoA wild-type, 0.6 mM pyruvate, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 9.2
-
 acetyl-CoA wild-type, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 9.8
-
 pyruvate wild-type, 1 microM isoleucine, 4 mM acetyl CoA, 0.8 mM MnCl2, 50 mM KCl, 15 nM enzyme, 0.1 M HEPES, pH 7.7 Leptospira interrogans
2.3.3.21 52
-
 pyruvate wild-type, in the presence of 0.004 mM isoleucine, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 98
-
 pyruvate wild-type, in the presence of 0.001 mM isoleucine, 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans
2.3.3.21 150
-
 pyruvate wild-type, in the presence of 0.8 mM MnCl2, 50 mM KCl, 15 nM CMS, in a total volume of 50 microL using 0.1 M Hepes, pH 7.7 Leptospira interrogans