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Specificity versus catalytic potency: The role of threonine 44 in Escherichia coli dihydrodipicolinate synthase mediated catalysis

Dobson, R.C.; Perugini, M.A.; Jameson, G.B.; Gerrard, J.A.; Biochimie 91, 1036-1044 (2009)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
4.3.3.7
mutant T44S expressed in dapA-deficient Escherichia coli AT997r-
Escherichia coli
Crystallization (Commentary)
EC Number
Crystallization
Organism
4.3.3.7
mutant T44S, crystals are isomorphous to those of the wild-type enzyme, no significant modification in its tertiary or quaternary structure from that of the wild-type enzyme
Escherichia coli
Engineering
EC Number
Amino acid exchange
Commentary
Organism
4.3.3.7
T44S
the active site is intact, returns much but not all activity likely due to the flexibility of Ser44. Increased flexibility in the active site, which appears to facilitate the binding/reaction of substrate analogues
Escherichia coli
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
4.3.3.7
L-lysine
no difference in its sensitivity or behaviour with respect to L-lysine when compared to the wild-type
Escherichia coli
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
4.3.3.7
0.13
-
L-aspartate 4-semialdehyde
mutant T44S
Escherichia coli
4.3.3.7
0.92
-
pyruvate
mutant T44S
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
mutant T44S, no pyruvate added to the crude extract prior to sonication, purified by heat shock and ion-exchange chromatography, 20.7fold with a yield of 123%
Escherichia coli
Specific Activity [micromol/min/mg]
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
4.3.3.7
0.72
-
crude extract of mutant T44S
Escherichia coli
4.3.3.7
14.52
-
20.7fold purified mutant T44S
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
-
702479
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
10.4
-
pyruvate
mutant T44S
Escherichia coli
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
4.3.3.7
mutant T44S expressed in dapA-deficient Escherichia coli AT997r-
Escherichia coli
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
4.3.3.7
mutant T44S, crystals are isomorphous to those of the wild-type enzyme, no significant modification in its tertiary or quaternary structure from that of the wild-type enzyme
Escherichia coli
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
4.3.3.7
T44S
the active site is intact, returns much but not all activity likely due to the flexibility of Ser44. Increased flexibility in the active site, which appears to facilitate the binding/reaction of substrate analogues
Escherichia coli
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
4.3.3.7
L-lysine
no difference in its sensitivity or behaviour with respect to L-lysine when compared to the wild-type
Escherichia coli
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
4.3.3.7
0.13
-
L-aspartate 4-semialdehyde
mutant T44S
Escherichia coli
4.3.3.7
0.92
-
pyruvate
mutant T44S
Escherichia coli
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
4.3.3.7
mutant T44S, no pyruvate added to the crude extract prior to sonication, purified by heat shock and ion-exchange chromatography, 20.7fold with a yield of 123%
Escherichia coli
Specific Activity [micromol/min/mg] (protein specific)
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
4.3.3.7
0.72
-
crude extract of mutant T44S
Escherichia coli
4.3.3.7
14.52
-
20.7fold purified mutant T44S
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
-
702479
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
10.4
-
pyruvate
mutant T44S
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
1.1
-
pyruvate
mutant T44S
Escherichia coli
4.3.3.7
81
-
L-aspartate 4-semialdehyde
mutant T44S
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
1.1
-
pyruvate
mutant T44S
Escherichia coli
4.3.3.7
81
-
L-aspartate 4-semialdehyde
mutant T44S
Escherichia coli