Information on EC 5.1.1.4 - proline racemase

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

EC NUMBER
COMMENTARY hide
5.1.1.4
-
RECOMMENDED NAME
GeneOntology No.
proline racemase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
L-proline = D-proline
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
isomerization
-
-
racemization
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Arginine and proline metabolism
-
-
L-ornithine degradation II (Stickland reaction)
-
-
Metabolic pathways
-
-
proline metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
proline racemase
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CAS REGISTRY NUMBER
COMMENTARY hide
9024-09-3
-
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
catalyzes step 8 in the ornithine fermentation pathway
physiological function
additional information
-
the enzyme is a two-base racemase
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
D-proline
L-proline
show the reaction diagram
L-azetidine-2-carboxylate
D-azetidine-2-carboxylate
show the reaction diagram
L-pipecolate
D-pipecolate
show the reaction diagram
L-Pro
D-Pro
show the reaction diagram
L-proline
D-proline
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
L-Pro
D-Pro
show the reaction diagram
-
overexpression of TcPRAC leads to an increase in parasite differentiation into infective forms and its subsequent penetration into host cells. During infection of its mammalian host, the parasite secretes a proline racemase that contributes to parasite immune evasion by acting as a B-cell mitogen
-
-
?
L-proline
D-proline
show the reaction diagram
additional information
?
-
-
a free and intact active site of the enzyme is necessary to allow mitogenicity
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
-
37 mM, 25% inhibition
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(E)-4-oxopent-2-enoic acid
-
an irreversible strong competitive inhibitor, which hampers Trypanosoma cruzi intracellular differentiation and fate in mammalian host cells
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(E)-5-bromo-4-oxopent-2-enoic acid
-
an irreversible strong competitive inhibitor, which hampers Trypanosoma cruzi intracellular differentiation and fate in mammalian host cells
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1,10-phenanthroline
-
-
1-carboxy-7-azoniabicyclo[2.2.1]heptane
-
a weak inhibitor
2-Furoic acid
-
weak
2-pyrrolecarboxylic acid
-
-
2-Thiophenecarboxylic acid
-
-
4-bromopyrazole-3-carboxylic acid
-
-
-
4-chloro-5-methyl-pyrazole-3-carboxylic acid
-
-
-
4-chloropyrazole-3-carboxylic acid
-
-
-
4-ethylpyrazole-3-carboxylic acid
-
-
-
7-azabicyclo[2.2.1]heptan-7-ium-1-carboxylate
-
a weak inhibitor of proline racemase, 29% inhibition at 142.5 mM
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alpha-Pipecoline
-
-
Bromoacetate
-
-
DELTA1-pyrroline-2-carboxylate
-
-
Fumaric acid
-
-
HPO42-
-
no inhibition by H2PO4-
iodoacetamide
iodoacetate
lipoic acid
-
weak
Maleic acid
malonic acid
-
-
Methoxyacetic acid
-
weak
Phthalic acid
-
-
pipecolic acid
-
weak
pyrazole-3-carboxylic acid
-
-
-
pyrrole-2-carboxylate
pyrrole-2-carboxylic acid
reduced glutathione
-
weak
succinic acid
-
-
tetrahydro-1H-pyrrolizine-7a(5H)-carboxylate
-
a bicyclic substrate-product analogue and noncompetitive inhibitor of proline racemase
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Tetrahydrofuroic acid
-
weak
thioglycolate
-
weak
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
NaBH4
-
activates
SH compounds
-
activate, e.g. 2-mercaptoethanol, 1,3-dimercaptoethanol
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.092 - 106
D-proline
1.21 - 154
L-proline
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0235 - 3.18
D-proline
0.092 - 14.15
L-proline
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0065 - 26.2
D-proline
0.0013 - 2.45
L-proline
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.006 - 0.01
2-pyrrolecarboxylic acid
-
recombinant His-tagged enzyme, pH 6.0, 37C
1
4-bromopyrazole-3-carboxylic acid
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recombinant His-tagged enzyme, pH 6.0, 37C
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2
4-chloro-5-methyl-pyrazole-3-carboxylic acid
-
recombinant His-tagged enzyme, pH 6.0, 37C
-
0.3
4-chloropyrazole-3-carboxylic acid
-
recombinant His-tagged enzyme, pH 6.0, 37C
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2
4-ethylpyrazole-3-carboxylic acid, pyrazole-3-carboxylic acid
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recombinant His-tagged enzyme, pH 6.0, 37C
-
0.0036 - 0.02
pyrrole-2-carboxylic acid
111
tetrahydro-1H-pyrrolizine-7a(5H)-carboxylate
-
pH and temperature not specified in the publication
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
349
1-carboxy-7-azoniabicyclo[2.2.1]heptane
Clostridium sticklandii
-
pH and temperature not specified in the publication
0.217 - 0.58
pyrrole-2-carboxylate
67
tetrahydro-1H-pyrrolizine-7a(5H)-carboxylate
Clostridium sticklandii
-
pH and temperature not specified in the publication
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.6 - 8.1
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pH 5.5: no detectable activity, rapid fall of activity below pH 6.5, activity is relatively stable between pH 6.7 and 8.1
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
24 - 37
-
at 37C the activity is 35-40% greater than at 24C
90 - 100
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5
-
isoelectric focusing
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
TcPRAC localizes in the cytoplasm of intracellular amastigote forms of the parasite, near the flagellar pocket of the parasites
Manually annotated by BRENDA team
-
membrane-bound and secreted forms of enzyme are present upon differentiation of the parasite into non-dividing infective forms
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Brucella melitensis biotype 1 (strain 16M / ATCC 23456 / NCTC 10094)
Trypanosoma cruzi (strain CL Brener)
Trypanosoma cruzi (strain CL Brener)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40000
recombinant enzyme, determined by SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
-
2 * 40100, TcPRACB, SDS-PAGE; 2 * 45800, TcPRACA, SDS-PAGE
homodimer
monomer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
best crystals are obtained by mixing 2-3 microliter of the protein solution with an equal volume of crystallization buffer (0.1 M ammonium acetate/50 mM tri-sodium citrate dihydrate, pH 5.6/15% w/v polyethylene glycol 4000), equilibrated over 1 ml of the same buffer; crystal structure analysis shows that TcPRACA is a homodimer, with each monomer folded in two symmetric alpha/beta subunits separated by a deep crevice. The structure of TcPRACA in complex with a transition-state analog, pyrrole-2-carboxylic acid, reveals the presence of one reaction center per monomer, with two Cys residues optimally located to perform acid/base catalysis through a carbanion stabilization mechanism
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70
-
half-life: 3.6 min, in D2O 17 min
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 50% glycerol or diluted in sodium acetate buffer, pH 6.0, activity is impaired
-
-20C, 50% glycerol or diluted in sodium acetate buffer, pH 6.0, stable
-
-20C, as ammonium sulfate precipitate, stable
-
23C, 0.5 M imidazole buffer, pH 8.0, 10 days, TcPRACA loses 84% of its activity
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23C, 0.5 M imidazole buffer, pH 8.0, 10 days, TcPRACB is stable, TcPRACA loses 84% of its activity
-
stored 6 months in the refrigerator without significant loss of activity
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2 forms of enzyme: one binds L-Pro and the other D-Pro
-
by affinity chromatography using nickel-nitrilotriacetic acid-agarose columns
cobalt metal-affinity resin column chromatography
-
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
-
recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
-
TcPRACA protein is purified with immobilized metal affinity chromatography on nickel columns. The active peak is further submitted to gel filtration chromatography at 2.5 ml/min in a Superdex200 26/60 column
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned in Escherichia coli as a 6xHis-tag fusion protein
expressed in Escherichia coli BL21(DE3) cells
-
expression in Escherichia coli
expression in Escherichia coli BL21 (DE3) with a C-terminal 6-His tag
recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
-
recombinant expression of His6-tagged enzyme in Escherichia coli strain BL21(DE3)
-
TcPRACA is expressed in Escherichia coli BL21 (DE3)
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the paralogous genes TcPRACA (A+) and TcPRACB (B++) are overexpressed in non-infective epimastigote forms using appropriate vectors to obtain stable chromosomal integration of these genes in sense and antisense orientations; the paralogous genes TcPRACA (A+) and TcPRACB (B++) are overexpressed in non-infective epimastigote forms using appropriate vectors to obtain stable chromosomal integration of these genes in sense and antisense orientations
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
2 paralogous copies of proline racemase genes are present per parasite haploid genome and they are differentially expressed during Trypanosoma cruzi development. Non-infective forms of the parasite expressing full length antisense TcPRACB RNA (functional knockdown) are not viable, whereas functional TcPRACA-knock down (A-) epimastigotes survive only poorly even under low selection pressure for recombinant parasites. Extracts from parasites overexpressing TcPRAC genes display more D-amino acid-containing peptides than wild type or knock down controls. The metacyclic form/epimastigote D-amino acid ratio obtained from parasites that developed in higher concentrations of proline (5.5) is greater than the metacyclic form/epimastigote D-amino acid ratio from wild type (2). Parasites overexpressing the intracytoplasmic isoform of proline racemase (B++) that has differentiated in 1x or 3x proline conditions display higher metacyclic form/epimastigote D-amino acid ratios than the respective wild type controls. Although they present a relative increase in metacyclic form/epimastigote D-amino acid ratios, overexpressors of the secreted version of TcPRAC (A+) show lower levels of D-amino acids than wild type or B++ parasites. Since TcPRACA transcripts appear to be more highly expressed at the end of metacyclogenesis, it is tempting to consider that racemisation of intracellular proline during this stage of development instead depends on the cytoplasmic version of the enzyme (TcPRACB); 2 paralogous copies of proline racemase genes are present per parasite haploid genome and they are differentially expressed during Trypanosoma cruzi development. Non-infective forms of the parasite expressing full length antisense TcPRACB RNA (functional knockdown) are not viable, whereas functional TcPRACA-knock down (A-) epimastigotes survive only poorly even under low selection pressure for recombinant parasites. Extracts from parasites overexpressing TcPRAC genes display more D-amino acid-containing peptides than wild type or knock down controls. The metacyclic form/epimastigote D-amino acid ratio obtained from parasites that developed in higher concentrations of proline (5.5) is greater than the metacyclic form/epimastigote D-amino acid ratio from wild type (2). Parasites overexpressing the intracytoplasmic isoform of proline racemase (B++) that has differentiated in 1x or 3x proline conditions display higher metacyclic form/epimastigote D-amino acid ratios than the respective wild type controls. Although they present a relative increase in metacyclic form/epimastigote D-amino acid ratios, overexpressors of the secreted version of TcPRAC (A+) show lower levels of D-amino acids than wild type or B++ parasites. Since TcPRACA transcripts appear to be more highly expressed at the end of metacyclogenesis, it is tempting to consider that racemisation of intracellular proline during this stage of development instead depends on the cytoplasmic version of the enzyme (TcPRACB)
in amastigote forms of the parasites, the intensity of anti-TcPRAC labeling varies according to the time post infection reaching the highest signal of TcPRAC expression after 48 h, while the number of intracellular parasites increases by amastigote multiplication
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recombinant epimastigote parasites overexpressing TcPRAC genes coding for proline racemase present an augmented ability to differentiate into metacyclic infective forms and subsequently penetrate host-cells in vitro
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
R240W
mutant enzyme shows similar kinetic constants for proline and hydroxyproline as compared to the wild-type enzyme
W241F the mutant enzyme
C130S
-
mutation of the catalytic Cys residues abolishes the enzymatic activity but preserves the mitogenic properties of the protein
C300S
-
mutation of the catalytic Cys residues abolishes the enzymatic activity but preserves the mitogenic properties of the protein
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
enzyme has potential to be used as a drug target for this parasite-based trypanosomiasis
medicine
-
potential of the enzyme as a critical target for drug development against Chagas disease. The enzyme is absent in mammalian host, suggesting that inhibition of proline racemase may have therapeutic potential; potential of the enzyme as a critical target for drug development against Chagas disease. The enzyme is absent in mammalian host, suggesting that inhibition of proline racemase may have therapeutic potential
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