Information on EC 5.1.1.10 - amino-acid racemase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
5.1.1.10
-
RECOMMENDED NAME
GeneOntology No.
amino-acid racemase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
an L-amino acid = a D-amino acid
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
racemization
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Cysteine and methionine metabolism
-
-
cysteine metabolism
-
-
D-Arginine and D-ornithine metabolism
-
-
D-Glutamine and D-glutamate metabolism
-
-
Glycine, serine and threonine metabolism
-
-
Metabolic pathways
-
-
serine metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
amino-acid racemase
A pyridoxal-phosphate protein.
CAS REGISTRY NUMBER
COMMENTARY hide
9068-61-5
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
bifunctional enzyme, racemization of serine and elimination of L-serine and L-serine-O-sulfate to form pyruvate
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-oxoglutarate + L-ornithine
D-glutamate + L-glutamate
show the reaction diagram
-
-
-
?
D-2,4-Diaminobutyrate
L-2,4-Diaminobutyrate
show the reaction diagram
-
10% of the activity relative to D-Lys
-
-
D-Ala
L-Ala
show the reaction diagram
12% activity, compared to D-Lys
-
-
?
D-Arg
L-Arg
show the reaction diagram
D-Asn
L-Asn
show the reaction diagram
-
-
-
-
r
D-Asp
L-Asp
show the reaction diagram
-
6% of the activity relative to D-Gln
-
-
D-aspartate
L-aspartate
show the reaction diagram
-
lower substrate affinity for D-aspartate compared to L-aspartate
-
-
?
D-ethionine
L-ethionine
show the reaction diagram
11% activity, compared to D-Lys
-
-
?
D-Gln
L-Gln
show the reaction diagram
-
D-Glu is the best substrate
-
-
D-homoarginine
L-homoarginine
show the reaction diagram
6.2% activity, compared to D-Lys
-
-
?
D-Lys
L-Lys
show the reaction diagram
100% activity
-
-
?
D-Met
L-Met
show the reaction diagram
D-norvaline
L-norvaline
show the reaction diagram
6.5% activity, compared to D-Lys
-
-
?
D-ornithine
L-ornithine
show the reaction diagram
42% activity, compared to D-Lys
-
-
?
D-Phe
L-Phe
show the reaction diagram
-
1% of the velocity relative to D-Glu
-
-
D-serine
L-serine
show the reaction diagram
D-serine
pyruvate + NH3
show the reaction diagram
-
alpha,beta-elimination reaction
-
-
?
L-2-Aminobutyrate
D-2-Aminobutyrate
show the reaction diagram
L-Ala
D-Ala
show the reaction diagram
L-Asn
D-Asn
show the reaction diagram
-
-
-
-
r
L-Asn
L-Asn
show the reaction diagram
L-aspartate
D-aspartate
show the reaction diagram
-
higher substrate affinity for L-aspartate compared to D-aspartate
-
-
?
L-Citrulline
D-Citrulline
show the reaction diagram
L-Ethionine
D-Ethionine
show the reaction diagram
L-Glu
D-Glu
show the reaction diagram
L-His
D-His
show the reaction diagram
L-Homoarginine
L-Homoarginine
show the reaction diagram
L-Homocitrulline
D-Homocitrulline
show the reaction diagram
L-Homocysteine
D-Homocysteine
show the reaction diagram
-
55% of the activity relative to L-Lys
-
-
L-Homoserine
L-Homoserine
show the reaction diagram
L-Leu
D-Leu
show the reaction diagram
L-Lys
D-Lys
show the reaction diagram
L-Met
D-Met
show the reaction diagram
L-Methionine
D-Methionine
show the reaction diagram
-
-
-
-
r
L-Norleucine
D-Norleucine
show the reaction diagram
L-Norvaline
D-Norvaline
show the reaction diagram
-
-
-
-
L-Orn
D-Orn
show the reaction diagram
L-Selenohomocysteine
D-Selenohomocysteine
show the reaction diagram
-
27% of the activity relative to L-Lys
-
-
L-Ser
D-Ser
show the reaction diagram
L-serine
D-serine
show the reaction diagram
L-serine
pyruvate + NH3
show the reaction diagram
-
alpha,beta-elimination reaction
-
-
?
L-serine
S-serine
show the reaction diagram
-
similar substrate affinity for both L-serine and D-serine
-
-
?
L-Thr
L-Thr
show the reaction diagram
L-threonine
3-hydroxy-2-butenoic acid + NH3
show the reaction diagram
-
alpha,beta-elimination reaction
-
-
?
L-Trp
D-Trp
show the reaction diagram
-
-
-
-
r
N-acetyl-D-alanine
N-acetyl-L-alanine
show the reaction diagram
-
-
-
-
r
N-acetyl-D-asparagine
N-acetyl-L-asparagine
show the reaction diagram
-
-
-
-
r
N-acetyl-D-methionine
N-acetyl-L-methionine
show the reaction diagram
-
-
-
-
r
N-acetyl-D-phenylalanine
N-acetyl-L-phenylalanine
show the reaction diagram
-
-
-
-
r
N-acetyl-D-tryptophan
N-acetyl-L-tryptophan
show the reaction diagram
-
-
-
-
r
N-carbamoyl-L-methionine
N-carbamoyl-D-methionine
show the reaction diagram
-
-
-
-
r
N-succinyl-L-alanine
N-succinyl-D-alanine
show the reaction diagram
-
-
-
-
r
N-succinyl-L-phenylalanine
N-succinyl-D-phenylalanine
show the reaction diagram
N6-Acetyl-L-Lys
N6-Acetyl-D-Lys
show the reaction diagram
S-Methyl-L-Cys
S-Methyl-D-Cys
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
pyridoxal 5'-phosphate
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ni2+
-
enhances activity
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3-chloroalanine
-
inactivator binds to the same Lys residue which binds pyridoxyl 5'-phosphate
3-Fluoroalanine
-
inactivator binds to the same Lys residue which binds pyridoxyl 5'-phosphate
8-hydroxy-5-quinolinesulfonic acid
-
-
aminooxyacetic acid
-
complete inhibition at 0.002 mM
ATP
-
inhibitory to L-serine O-sulfate dehydration reaction, activating for racemization reraction
beta-mercaptoethanol
-
-
Co2+
-
inhibition of both activities
Cu2+
-
inhibition of both activities
D-cycloserine
-
-
D-penicillamine
-
-
dithiothreitol
-
-
EDTA
-
inhibition of both activities
Fe2+
-
slight inhibition of both activities
hydroxylamine
Ni2+
-
slight inhibition of both activities
O-acetylserine
S-(N-Methylthiocarbamoyl)-D-Cys
-
-
S-(N-Methylthiocarbamoyl)-L-Cys
-
-
Zn2+
-
inhibition of both activities
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
7.4
D-Ala
-
-
18 - 103
D-Asn
94
D-Aspartate
-
in 50 mM Tris-HCl buffer, pH 8.0, at 30C
7.8
D-Gln
-
-
0.42 - 30
D-Lys
1.6 - 42
D-Met
4.8 - 24.3
D-methionine
25.7 - 26
D-Phe
1 - 18
D-Ser
14.5 - 49
D-serine
9.8
D-Thr
-
-
36
L-2-aminobutyrate
7
L-Ala
-
-
30 - 101
L-Asn
8
L-aspartate
-
in 50 mM Tris-HCl buffer, pH 8.0, at 30C
35
L-ethionine
-
-
33
L-Leu
0.55 - 2.5
L-Lys
1.2 - 39
L-Met
4.1 - 22.3
L-methionine
0.9
L-Orn
3.8 - 30
L-serine
31
L-Thr
-
-
17
N-acetyl-D-alanine
-
-
18
N-acetyl-D-asparagine
-
-
7
N-acetyl-D-methionine
-
-
2 - 3
N-acetyl-D-phenylalanine
-
-
2
N-acetyl-D-tryptophan
-
-
41
N-acetyl-L-alanine
-
-
27
N-acetyl-L-asparagine
-
-
8
N-acetyl-L-methionine
-
-
43
N-acetyl-L-phenylalanine
-
-
2
N-acetyl-L-tryptophan
5
N-carbamoyl-L-methionine
-
-
0.13
N-succinyl-D-alanine
-
-
0.04
N-succinyl-D-phenylalanine
-
-
2.6
N-Succinyl-L-Ala
-
-
0.12
N-succinyl-L-alanine
-
-
3.8
N-Succinyl-L-His
-
-
3.7
N-Succinyl-L-Ile
-
-
1.3
N-Succinyl-L-Leu
-
-
3.6
N-Succinyl-L-Met
-
-
0.8
N-succinyl-L-Phe
-
-
0.13
N-succinyl-L-phenylalanine
-
-
3
N-Succinyl-L-Ser
-
-
7.1
N-Succinyl-L-Trp
-
-
2.6
N-Succinyl-L-Tyr
-
-
2.5
N-Succinyl-L-Val
-
-
additional information
N-Succinyl-L-Arg
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
14.5
D-serine
Mus musculus
-
pH 8.0, 37C, presence of 1 mM ATP, racemization reaction
3.8
L-serine
Mus musculus
-
pH 8.0, 37C, presence of 1 mM ATP, racemization reaction
0.8
N-acetyl-D-alanine
Geobacillus kaustophilus
-
-
0.07
N-acetyl-D-asparagine
Geobacillus kaustophilus
-
-
20
N-acetyl-D-methionine
Geobacillus kaustophilus
-
-
10
N-acetyl-D-phenylalanine
Geobacillus kaustophilus
-
-
0.09
N-acetyl-D-tryptophan
Geobacillus kaustophilus
-
-
2
N-acetyl-L-alanine
Geobacillus kaustophilus
-
-
0.06
N-acetyl-L-asparagine
Geobacillus kaustophilus
-
-
22
N-acetyl-L-methionine
Geobacillus kaustophilus
-
-
16
N-acetyl-L-phenylalanine
Geobacillus kaustophilus
-
-
0.15
N-acetyl-L-tryptophan
Geobacillus kaustophilus
-
-
2
N-carbamoyl-D-methionine
15
N-succinyl-D-alanine
Geobacillus kaustophilus
-
-
2
N-succinyl-D-phenylalanine
Geobacillus kaustophilus
-
-
89
N-Succinyl-L-Ala
Geobacillus kaustophilus
-
-
43
N-succinyl-L-alanine
Geobacillus kaustophilus
-
-
0.014
N-Succinyl-L-Arg
Geobacillus kaustophilus
-
-
2.5
N-Succinyl-L-Asn
Geobacillus kaustophilus
-
-
0.071
N-Succinyl-L-Asp
Geobacillus kaustophilus
-
-
9
N-Succinyl-L-Gln
Geobacillus kaustophilus
-
-
0.073
N-Succinyl-L-Glu
Geobacillus kaustophilus
-
-
2
N-Succinyl-L-His
Geobacillus kaustophilus
-
-
35
N-Succinyl-L-Ile
Geobacillus kaustophilus
-
-
17
N-Succinyl-L-Leu
Geobacillus kaustophilus
-
-
2.1
N-Succinyl-L-Lys
Geobacillus kaustophilus
-
-
53
N-Succinyl-L-Met
Geobacillus kaustophilus
-
-
19
N-succinyl-L-Phe
Geobacillus kaustophilus
-
-
5
N-succinyl-L-phenylalanine
Geobacillus kaustophilus
-
-
45
N-Succinyl-L-Ser
Geobacillus kaustophilus
-
-
7.9
N-Succinyl-L-Trp
Geobacillus kaustophilus
-
-
52
N-Succinyl-L-Tyr
Geobacillus kaustophilus
-
-
92
N-Succinyl-L-Val
Geobacillus kaustophilus
-
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.047
N-acetyl-D-alanine
Geobacillus kaustophilus
-
-
15855
0.004
N-acetyl-D-asparagine
Geobacillus kaustophilus
-
-
20393
3
N-acetyl-D-methionine
Geobacillus kaustophilus
-
-
10717
0.467
N-acetyl-D-phenylalanine
Geobacillus kaustophilus
-
-
15857
0.061
N-acetyl-D-tryptophan
Geobacillus kaustophilus
-
-
16118
0.039
N-acetyl-L-alanine
Geobacillus kaustophilus
-
-
2972
0.002
N-acetyl-L-asparagine
Geobacillus kaustophilus
-
-
6135
2.8
N-acetyl-L-methionine
Geobacillus kaustophilus
-
-
1165
0.37
N-acetyl-L-phenylalanine
Geobacillus kaustophilus
-
-
2855
0.06
N-acetyl-L-tryptophan
Geobacillus kaustophilus
-
-
4835
1.2
N-carbamoyl-D-methionine
Geobacillus kaustophilus
-
-
16690
1.1
N-carbamoyl-L-methionine
Geobacillus kaustophilus
-
-
4105
119
N-succinyl-D-alanine
Geobacillus kaustophilus
-
-
40130
38.1
N-succinyl-D-phenylalanine
Geobacillus kaustophilus
-
-
40131
352
N-succinyl-L-alanine
Geobacillus kaustophilus
-
-
40129
35.1
N-succinyl-L-phenylalanine
Geobacillus kaustophilus
-
-
14015
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00588
-
substrate Trp, wildtype BAR
0.00706
-
substrate Phe, mutant Y396C
0.00782
-
substrate Ala, wildtype BAR
0.00838
-
substrate Phe, wildtype BAR
0.0121
-
substrate Ala, mutant I83L, D361V, Y396C
0.0141
-
substrate Ala, mutant Y293A, Y301S, Y396C
0.0144
-
substrate Ala, mutant L126H, Y396C
0.0249
-
substrate Phe, mutant Y293A, Y301S, Y396C
0.0257
-
substrate Phe, mutant I83L, D361V, Y396C
0.026
-
substrate Ala, mutant I384M
0.0267
-
substrate Trp, mutant Y293A, Y301S, Y396C
0.0303
-
substrate Trp, mutant I83L, D361V, Y396C
0.03215
-
substrate Lys, mutant Y396C
0.0436
-
substrate Phe, mutant L126H, Y396C
0.0487
-
substrate Trp, mutant Y396C
0.0524
-
substrate Trp, mutant L126H, Y396C
0.0744
-
substrate Phe, mutant I384M
0.124
-
substrate Trp, mutant I384M
0.132
-
substrate Lys, wildtype BAR
0.223
-
substrate Lys, mutant I384M
0.458
-
substrate Lys, mutant Y293A, Y301S, Y396C
0.473
-
substrate Lys, mutant I83L, D361V, Y396C
0.477
-
substrate Lys, mutant L126H, Y396C
0.825
-
substrate Ala, mutant Y396C
920
-
L-Lys as substrate
1120
recombinant protein
1250
-
L-Lys as substrate
additional information
-
wildtype BAR, specific activity for L-Lys 100%, L-Arg 65%, L-Ala 33%, L-Ser 20%, L-Met 14%, L-Cys 14%, L-Leu 3.3%, L-His 1.6%, L-Phe 0.29%, L-Pro 0.1.3%, L-Thr 0.069%, L-Asn 0.054%, L-Asp 0.01%, L-Trp 0.006%, L-Val 0.003%
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5 - 9
-
-
7.5 - 10
-
-
7.5 - 8.5
-
-
8 - 9.5
-
-
8.5
-
assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5
-
negligible activity below
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20
-
assay at
50 - 60
-
-
65
maximum activity
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
DAR1 is localized to the medial region of the cerebral ganglion where the F- and C-clusters are situated
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
41500
calculated from cDNA
42300
-
molecular weight of monomeric subunit, SDS-PAGE
43000
-
SDS-PAGE
45000
-
SDS-PAGE
55000
-
isoforms A and B, gel filtration
57000
-
gel filtration
62000 - 65000
-
gel filtration
73000
-
gel filtration
76000
-
sedimentation equilibrium method
78000
-
gel filtration
82000
-
gel filtration
84000
gel filtration
110000
150000
-
gel filtration, tetramer
170000
-
tetramer, analytical ultracentrifugation
177300
-
tetramer, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
homotetramer
-
4 * 43000, SDS-PAGE
tetramer
-
gel filtration and analytical ultracentrifugation. Tetrameric structure in the absence or presence of Co2+, 4 * 42300 Da
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
N-terminal signal sequence is cleaved off
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 8.5
-
the enzyme shows little racemase activity below pH 6.5 and an increasing activity between pH 7.0-8.5 in 50 mM Tris-HCl buffer
715591
7 - 11
-
at 30C, 1 h, stable
2049
10
-
at 50C, 10 min, stable
2049
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
14 - 50
-
the enzyme activity increases between 14-45C and starts to decrease at 50C for both the Ser and Asp conversion
50
-
pH 10.0, 10 min, stable
80
-
pH 10.0, 10 min, most of the activity is lost
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
buffer without organic content provides excellent stability at moderate temperatures (2035C) while addition of 20% acetonitrile or methanol drastically reduces the half-life of the racemase
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
0-5C, 0.01 M potassium phosphate buffer, pH 7.0, 2 mM pyridoxal 5'-phosphate, 1 month, stable
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
both isoforms A and B
-
expression in Escherichia coli with N-terminal His-tag, purification protocol from inclusion bodies
-
His-tag is removed by thrombin cleavage
-
His6-tagged BAR is applied onto a His-Trap HP 1-ml column
-
Ni-NTA column chromatography and Sepharcryl 200 gel filtration
-
protein is purified by applying to a diethylaminoethyl sepharose fast flow column and a phenyl sepharose 6 fast flow column
-
purified in a one-step procedure by immobilized cobalt affinity chromatography
-
recombinant enzyme expressed in insect cells
-
two enzyme lyophilisates of different purity are obtained from which the crude is sufficient for the racemization of methionine and the pure is used for asparagine
-
using Ni-NTA chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
amino acid racemase of Pseudomonas putida DSM 3263 is overexpressed in Escherichia coli and delivered cell free extract with easily sufficient activity (2050 U/mg total protein) for application in an enzyme membrane reactor (EMR) setting
-
expressed as a His-taged fusion protein
-
expressed in Escherichia coli as a His-tagged fusion protein
expressed in Escherichia coli BL21(DE3) cells
-
over-expressed in Escherichia coli
protein is expressed in Escherichia coli strain BL21
-
recombinant BAR is cloned in Escherichia coli BL-21 as His-tagged BAR
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H152S
-
ratio of elimination reaction to racemization is 1.4 compared to 3.7 in wild-type
N154F
-
ratio of elimination reaction to racemization is 0.33 compared to 3.7 in wild-type
P153S
-
ratio of elimination reaction to racemization is 0.24 compared to 3.7 in wild-type
Q155D
-
ratio of elimination reaction to racemization is 0.25 compared to 3.7 in wild-type
I384M
-
I384M mutant BAR shows the highest activity for Trp. Using I384M mutant BAR the proportion of D-Trp reaches 43% while wildtype BAR racemizes only 6% of initial L-Trp; specific activity: 0.124 (substrate Trp), 0.0744 (substrate Phe), 0.223 (substrate Lys), 0.026 (substrate Ala)
I83L/D361V/Y396C
-
specific activity: 0.0303 (substrate Trp), 0.0257 (substrate Phe), 0.473 (substrate Lys), 0.0121 (substrate Ala)
L126H/Y396C
-
specific activity: 0.0524 (substrate Trp), 0.0436 (substrate Phe), 0.477 (substrate Lys), 0.0144 (substrate Ala)
Y293A/Y301S/Y396C
-
specific activity: 0.0267 (substrate Trp), 0.0249 (substrate Phe), 0.458 (substrate Lys), 0.0141 (substrate Ala)
Y396C
-
specific activity: 0.0487 (substrate Trp), 0.00706 (substrate Phe), 0.03215 (substrate Lys), 0.825 (substrate Ala)
additional information
-
random mutagenesis on bar gene is performed to obtain mutant BAR derivates with high activity for Trp. Five positive mutant are isolated after the two-step-screening of the randomly mutated BAR; substitutions at Y396 and I384 increases the Trp specific racemization activity and the racemazation activity for overall amino acids, respectively
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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simple procedure for in situ analysis of stereospecificity of C-4 hydrogen transfer of NADH by an NAD-dependent dehydrogenase by combination with amino acid racemase, EC 5.1.1.10, and L-leucine dehydrogenase, EC 1.4.1.9
biotechnology
synthesis
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enzymatic production of L-Trp from DL-Ser and indole by a coupled reaction of tryptophan synthase and amino acid racemase