BRENDA - Enzyme Database show

How essential is the essential active-site lysine in dihydrodipicolinate synthase?

Soares da Costa, T.P.; Muscroft-Taylor, A.C.; Dobson, R.C.; Devenish, S.R.; Jameson, G.B.; Gerrard, J.A.; Biochimie 92, 837-845 (2010)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
4.3.3.7
DHDPS overexpressed in Escherichia coli AT997recA-, transformed with site-directed mutants based on the pBluescript plasmid pJG001
Escherichia coli
Crystallization (Commentary)
EC Number
Crystallization
Organism
4.3.3.7
by the hanging drop-vapour diffusion method, mutants K161A and K161R solved at resolutions of 2.0 and 2.1 A, respectively. They show no changes in their secondary or tertiary structures when compared to the wild-type structure. Crystal structure of mutant K161A with pyruvate bound at the active site solved at a resolution of 2.3 A, reveals a defined binding pocket for pyruvate that is thus not dependent upon lysine 161
Escherichia coli
Engineering
EC Number
Amino acid exchange
Commentary
Organism
4.3.3.7
K161A
catalytically active, significant decrease in activity. Is not inactivated when incubated with pyruvate and the reducing agent sodium borohydride. Negligible heat production associated with pyruvate binding to the mutant enzyme, consistent with the lack of Schiff base formation
Escherichia coli
4.3.3.7
K161R
catalytically active, significant decrease in activity. Is not inactivated when incubated with pyruvate and the reducing agent sodium borohydride. Negligible heat production associated with pyruvate binding to the mutant enzyme, consistent with the lack of Schiff base formation
Escherichia coli
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
4.3.3.7
lysine
inhibition of wild-type DHDPS by lysine with respect to pyruvate is partial and uncompetitive, and partial non-competitive with respect to L-aspartate 4-semialdehyde. Ethanolamine, n-butylamine, 1-amino-2-propanol, 3-amino-1-propanol, iso-butylamine and Tris-HCl cannot rescue activity
Escherichia coli
4.3.3.7
Sodium borohydride
wild-type DHDPS is inactivated when incubated with pyruvate, whereas incubation with L-aspartate 4-semialdehyde has no effect
Escherichia coli
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
4.3.3.7
0.12
-
L-aspartate 4-semialdehyde
mutant K161R, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR; wild-type, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
0.15
-
pyruvate
wild-type, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
0.23
-
L-aspartate 4-semialdehyde
mutant K161A, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
0.45
-
pyruvate
mutant K161A, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
0.57
-
pyruvate
mutant K161R, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
4.3.3.7
Escherichia coli
P0A6L2
-
-
Purification (Commentary)
EC Number
Commentary
Organism
4.3.3.7
wild-type and mutants, by gel filtration
Escherichia coli
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
4.3.3.7
L-aspartate 4-semialdehyde + pyruvate
condensation reaction between both substrates via the formation of a Schiff base intermediate between pyruvate and the absolutely conserved active-site lysine. Although lysine 161 is important in the wild-type DHDPS-catalysed reaction, it is not absolutely essential for catalysis
702489
Escherichia coli
dihydrodipicolinate + 2 H2O
-
-
-
?
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
4.3.3.7
0.06
-
pyruvate
mutant K161A, at 30°C, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
0.16
-
pyruvate
mutant K161R, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
45
-
pyruvate
wild-type, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
Ki Value [mM]
EC Number
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
4.3.3.7
0.12
-
lysine
wild-type, with pyruvate as substrate
Escherichia coli
4.3.3.7
0.14
-
lysine
mutant K161A, with pyruvate as substrate; mutant K161R, with L-aspartate 4-semialdehyde as substrate; mutant K161R, with pyruvate as substrate
Escherichia coli
4.3.3.7
0.18
-
lysine
wild-type, with L-aspartate 4-semialdehyde as substrate
Escherichia coli
4.3.3.7
0.23
-
lysine
mutant K161A, with L-aspartate 4-semialdehyde as substrate
Escherichia coli
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
4.3.3.7
DHDPS overexpressed in Escherichia coli AT997recA-, transformed with site-directed mutants based on the pBluescript plasmid pJG001
Escherichia coli
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
4.3.3.7
by the hanging drop-vapour diffusion method, mutants K161A and K161R solved at resolutions of 2.0 and 2.1 A, respectively. They show no changes in their secondary or tertiary structures when compared to the wild-type structure. Crystal structure of mutant K161A with pyruvate bound at the active site solved at a resolution of 2.3 A, reveals a defined binding pocket for pyruvate that is thus not dependent upon lysine 161
Escherichia coli
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
4.3.3.7
K161A
catalytically active, significant decrease in activity. Is not inactivated when incubated with pyruvate and the reducing agent sodium borohydride. Negligible heat production associated with pyruvate binding to the mutant enzyme, consistent with the lack of Schiff base formation
Escherichia coli
4.3.3.7
K161R
catalytically active, significant decrease in activity. Is not inactivated when incubated with pyruvate and the reducing agent sodium borohydride. Negligible heat production associated with pyruvate binding to the mutant enzyme, consistent with the lack of Schiff base formation
Escherichia coli
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
4.3.3.7
lysine
inhibition of wild-type DHDPS by lysine with respect to pyruvate is partial and uncompetitive, and partial non-competitive with respect to L-aspartate 4-semialdehyde. Ethanolamine, n-butylamine, 1-amino-2-propanol, 3-amino-1-propanol, iso-butylamine and Tris-HCl cannot rescue activity
Escherichia coli
4.3.3.7
Sodium borohydride
wild-type DHDPS is inactivated when incubated with pyruvate, whereas incubation with L-aspartate 4-semialdehyde has no effect
Escherichia coli
Ki Value [mM] (protein specific)
EC Number
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
4.3.3.7
0.12
-
lysine
wild-type, with pyruvate as substrate
Escherichia coli
4.3.3.7
0.14
-
lysine
mutant K161A, with pyruvate as substrate; mutant K161R, with L-aspartate 4-semialdehyde as substrate; mutant K161R, with pyruvate as substrate
Escherichia coli
4.3.3.7
0.18
-
lysine
wild-type, with L-aspartate 4-semialdehyde as substrate
Escherichia coli
4.3.3.7
0.23
-
lysine
mutant K161A, with L-aspartate 4-semialdehyde as substrate
Escherichia coli
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
4.3.3.7
0.12
-
L-aspartate 4-semialdehyde
mutant K161R, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR; wild-type, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
0.15
-
pyruvate
wild-type, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
0.23
-
L-aspartate 4-semialdehyde
mutant K161A, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
0.45
-
pyruvate
mutant K161A, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
0.57
-
pyruvate
mutant K161R, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
4.3.3.7
wild-type and mutants, by gel filtration
Escherichia coli
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
4.3.3.7
L-aspartate 4-semialdehyde + pyruvate
condensation reaction between both substrates via the formation of a Schiff base intermediate between pyruvate and the absolutely conserved active-site lysine. Although lysine 161 is important in the wild-type DHDPS-catalysed reaction, it is not absolutely essential for catalysis
702489
Escherichia coli
dihydrodipicolinate + 2 H2O
-
-
-
?
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
4.3.3.7
0.06
-
pyruvate
mutant K161A, at 30°C, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
0.16
-
pyruvate
mutant K161R, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
45
-
pyruvate
wild-type, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
KCat/KM [mM/s]
EC Number
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
4.3.3.7
0.13
-
pyruvate
mutant K161A, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
0.26
-
L-aspartate 4-semialdehyde
mutant K161A, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
0.28
-
pyruvate
mutant K161R, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
1.3
-
L-aspartate 4-semialdehyde
mutant K161R, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
300
-
pyruvate
wild-type, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
380
-
L-aspartate 4-semialdehyde
wild-type, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
KCat/KM [mM/s] (protein specific)
EC Number
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
4.3.3.7
0.13
-
pyruvate
mutant K161A, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
0.26
-
L-aspartate 4-semialdehyde
mutant K161A, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
0.28
-
pyruvate
mutant K161R, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
1.3
-
L-aspartate 4-semialdehyde
mutant K161R, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
300
-
pyruvate
wild-type, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli
4.3.3.7
380
-
L-aspartate 4-semialdehyde
wild-type, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR
Escherichia coli