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Literature summary for 5.3.3.1 extracted from

  • Nam, G.H.; Kim, D.H.; Ha, N.C.; Jang do, S.; Yun, Y.S.; Hong, B.H.; Oh, B.H.; Choi, K.Y.
    Contribution of conserved amino acids at the dimeric interface to the conformational stability and the structural integrity of the active site in ketosteroid isomerase from Pseudomonas putida biotype B (2003), J. Biochem., 134, 101-110.
    View publication on PubMed

Crystallization (Commentary)

Crystallization (Comment) Organism
crystal structure of the R72A mutant enzyme determined at 2.5 A resolution belongs to the space group C2221 with cell dimensions of a = 36.37 A, b = 74.44 A and c = 96.06 A. Crystals are grown from a solution containing 2.0 M ammonioum acetate and 0.1 M sodium acetate at pH 4.6 by hanging drop vapor-diffusion method at 22°C Pseudomonas putida

Protein Variants

Protein Variants Comment Organism
E118A the free-energy change for unfolding in the absence of urea at 25°C is decreased by about 3.9 kcal/mol compared to wild-type value. Mutation increasex the dissociation constant for (+)-equilenin, a reaction intermediate analogue. 50% of the protein is unfolded at 4.46 M urea compared to 5.22 M for the wild-type enzyme. The turnover-number for 5-androstene-3,17-dione is 33% of that of the wild-type enzyme, the Km-value is 348% of that of the wild-type enzyme Pseudomonas putida
E118A/N120A the free-energy change for unfolding in the absence of urea at 25°C is decreased by about 9.5 kcal/mol compared to wild-type value. Mutation increases the dissociation constant for (+)-equilenin, a reaction intermediate analogue. 50% of the protein is unfolded at 3.89 M urea compared to 5.22 M for the wild-type enzyme. The turnover-number for 5-androstene-3,17-dione is 6% of that of the wild-type enzyme, the Km-value is 523% of that of the wild-type enzyme Pseudomonas putida
N120A the free-energy change for unfolding in the absence of urea at 25°C is decreased by about 7.8 kcal/mol compared to wild-type value. Mutation increasex the dissociation constant for (+)-equilenin, a reaction intermediate analogue. 50% of the protein is unfolded at 3.95 M urea compared to 5.22 M for the wild-type enzyme. The turnover-number for 5-androstene-3,17-dione is 17% of that of the wild-type enzyme, the Km-value is 516% of that of the wild-type enzyme Pseudomonas putida
R72A the free-energy change for unfolding in the absence of urea at 25°C is decreased by about 3.8 kcal/mol compared to wild-type value. Mutation increasex the dissociation constant for (+)-equilenin, a reaction intermediate analogue. 50% of the protein is unfolded at 4.74 M urea compared to 5.22 M for the wild-type enzyme. The turnover-number for 5-androstene-3,17-dione is 23% of that of the wild-type enzyme Pseudomonas putida

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0499
-
5-androstene-3,17-dione pH 7.0, 25°C, wild-type enzyme Pseudomonas putida
0.173
-
5-androstene-3,17-dione pH 7.0, 25°C, mutant enzyme E118A Pseudomonas putida
0.258
-
5-androstene-3,17-dione pH 7.0, 25°C, mutant enzyme N120A Pseudomonas putida
0.261
-
5-androstene-3,17-dione pH 7.0, 25°C, mutant enzyme E118A/N120A Pseudomonas putida

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
14000
-
2 * 14000, mutant enzymnes R72A, E118A, N120A and E18A/N120A, SDS-PAGE Pseudomonas putida

Organism

Organism UniProt Comment Textmining
Pseudomonas putida
-
biotype B
-

Renatured (Commentary)

Renatured (Comment) Organism
50% of the wild-type enzyme is unfolded at 5.22 M urea. Mutant enzymes R72A, E118A, N120A and E118A/N120A are unfolded at lower concentrations Pseudomonas putida

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
5-Androstene-3,17-dione
-
Pseudomonas putida 4-Androstene-3,17-dione
-
?

Subunits

Subunits Comment Organism
dimer 2 * 14000, mutant enzymnes R72A, E118A, N120A and E18A/N120A, SDS-PAGE Pseudomonas putida

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
25
-
the free-energy change for unfolding in the absence of urea at 25°C is 24.4 kcal/mol for the wild-type enzyme Pseudomonas putida

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1180
-
5-androstene-3,17-dione pH 7.0, 25°C, mutant enzyme E118A/N120A Pseudomonas putida
3710
-
5-androstene-3,17-dione pH 7.0, 25°C, mutant enzyme N120A Pseudomonas putida
4870
-
5-androstene-3,17-dione pH 7.0, 25°C, mutant enzyme R72A Pseudomonas putida
6920
-
5-androstene-3,17-dione pH 7.0, 25°C, mutant enzyme E118A Pseudomonas putida
21200
-
5-androstene-3,17-dione pH 7.0, 25°C, wild-type enzyme Pseudomonas putida