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Information on EC 3.4.17.11 - glutamate carboxypeptidase and Organism(s) Pseudomonas sp. and UniProt Accession P06621
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EC Tree
3.4.17.11 glutamate carboxypeptidaseSpecify your search results
Word Map
- 3.4.17.11
- methotrexate
-
prodrugs
-
high-dose
-
antibody-directed
- leucovorin
-
hdmtx
-
methotrexate-induced
-
gene-directed
- naaladase
-
gdept
-
antibody-enzyme
- medicine
-
anti-cea
-
anti-carcinoembryonic
-
prodrug-activating
- gcpii
- 2-pmpa
-
n-acetyl-aspartyl-glutamate
-
dampa
- drug development
- diagnostics
- pharmacology
The taxonomic range for the selected organisms is: Pseudomonas sp.
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Reaction Schemes
release of C-terminal glutamate residues from a wide range of N-acylating moieties, including peptidyl, aminoacyl, benzoyl, benzyloxycarbonyl, folyl and pteroyl groups
Synonyms
glucarpidase, carboxypeptidase g2, cpdg2, glutamate carboxypeptidase, carboxypeptidase g1, carboxypeptidase g, glutamyl carboxypeptidase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
carboxypeptidase G
-
-
-
-
carboxypeptidase G1
-
-
-
-
carboxypeptidase G2
folate hydrolase G2
-
-
-
-
glutamate carboxypeptidase
-
-
-
-
glutamyl carboxypeptidase
-
-
-
-
N-pteroyl-L-glutamate hydrolase
-
-
-
-
pteroylmonoglutamic acid hydrolase G2
-
-
-
-
carboxypeptidase G2
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hydrolysis of peptide bond
hydrolysis of peptide bond
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
9074-87-7
not distinguishable from EC 3.4.19.9 in Chemical Abstracts
94894-32-3
Pseudomonas isoenzyme 2
94894-33-4
Pseudomonas isoenzyme 2 precursor
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
3,5-difluorobenzoyl-L-glutamate + H2O
3,5-difluorobenzoate + L-glutamate
-
chemical exchange saturation transfer magnetic resonance study, i.e. CEST-MR to monitor the release of glutamate. CEST-MR affords the detection of CPG2 activity in vitro and supports the translation of CEST-MRI to assess CPG2-based gene therapy in vivo
-
?
methotrexate + H2O
4-amino-4-deoxy-N10-methylpteroic acid + L-glutamate
-
-
-
?
(2-chloroethyl)-(2-(methylsulfoyloxyethyl)amino)aminobenzyl-L-glutamic acid + H2O
(2-chloroethyl)-(2-(methylsulfoyloxyethyl)amino)aminobenzoic acid + L-glutamate
-
a prodrug
-
-
?
(4-[bis(2-bromoethyl)amino]-2,3,5-trifluorobenzoyl)-L-glutamic acid + H2O
4-[bis(2-bromoethyl)amino]-2,3,5-trifluorobenzoic acid + L-glutamate
-
a trifluorinated prodrug
-
-
?
(4-[bis(2-bromoethyl)amino]-3,5-difluorobenzoyl)-L-glutamic acid + H2O
4-[bis(2-bromoethyl)amino]-3,5-difluorobenzoic acid + L-glutamate
-
a difluorinated prodrug
-
-
?
(4-[bis(2-chloroethyl)amino]-2,3,5-trifluorobenzoyl)-L-glutamic acid + H2O
4-[bis(2-chloroethyl)amino]-2,3,5-trifluorobenzoic acid + L-glutamate
-
a trifluorinated prodrug
-
-
?
(4-[bis(2-chloroethyl)amino]-3,5-difluorobenzoyl)-L-glutamic acid + H2O
4-[bis(2-chloroethyl)amino]-3,5-difluorobenzoic acid + L-glutamate
-
a difluorinated prodrug
-
-
?
(4-[bis(2-iodoethyl)amino]-2,3,5-trifluorobenzoyl)-L-glutamic acid + H2O
4-[bis(2-iodoethyl)amino]-2,3,5-trifluorobenzoic acid + L-glutamate
-
a trifluorinated prodrug
-
-
?
(4-[bis(2-iodoethyl)amino]-3,5-difluorobenzoyl)-L-glutamic acid + H2O
4-[bis(2-iodoethyl)amino]-3,5-difluorobenzoic acid + L-glutamate
-
a difluorinated prodrug
-
-
?
(6R,S)-leucovorin + H2O
N-(6R,S)-5-formyl-5,6,7,8-tetrahydropteroic acid + L-glutamate
-
-
-
-
?
3,5-difluoro-4-[bis(2-iodoethyl)amino]benzoic acid + H2O
?
-
cognate drug
-
-
?
3,5-difluoro-4-[bis(2-iodoethyl)amino]benzoyl-L-glutamic acid + H2O
?
-
prodrug activated by CPG2
-
-
?
3,5-difluorobenzoyl-L-glutamic acid + H2O
3,5-difluorobenzoic acid + L-glutamate
-
-
-
-
?
3-fluoro-4-[bis(2-chloroethyl)amino]benzoyl-L-glutamic acid + H2O
?
-
prodrug activated by CPG2
-
-
?
4-[(2-chloroethyl)(2-methylsulphonyloxyethyl)amino]benzoic acid + H2O
?
-
cognate drug
-
-
?
4-[(2-chloroethyl)-(2-methylsulphonyloxyethyl)amino]benzoyl-L-glutamic acid + H2O
?
-
prodrug activated by CPG2
-
-
?
4-[bis(2-iodoethyl)-amino]-phenyloxycarbonyl-L-glutamic acid + H2O
?
-
prodrug activated by CPG2
-
-
?
di-tert-butyl 4-[[2'-[N,N-bis(2''-chloroethyl)amino]phenyl]carbamoyloxymethyl]phenyloxycarbonyl-L-glutamic acid + H2O
?
-
-
-
-
?
diallyl 4-[N-[4'-bis(2''-chloroethyl)aminophenyl]-N-methylcarbamoyloxymethyl]phenylcarbamoyl-L-glutamic acid + H2O
?
-
-
-
-
?
diallyl 4-[[2'-[N,N-bis(2''-chloroethyl)amino]phenyl]carbamoyloxymethyl]phenylcarbamoyl-L-glutamic acid + H2O
?
-
-
-
-
?
methotrexate + H2O
4-[(2,4-diamino-6-(pteridinyl)methyl)-methylamino]-benzoic acid + L-glutamate
5-formyltetrahydrofolic acid + H2O
5-formylpteroate + Glu
-
i.e. leucovorin
-
-
?
diethylstilbestrol-glutamate + H2O
diethylstilbestrol + L-glutamate
-
-
-
-
?
diethylstilbestrol-glutamate + H2O
diethylstilbestrol + L-glutamate
-
the water soluble substrate can be used as a prodrug that is hydrolyzed to yield the active drug in an antibody directed enzyme prodrug therapy
-
-
?
methotrexate + H2O
4-amino-4-deoxy-N10-methylpteroic acid + L-glutamate
-
-
-
?
methotrexate + H2O
4-amino-4-deoxy-N10-methylpteroic acid + L-glutamate
-
-
-
-
?
methotrexate + H2O
4-[(2,4-diamino-6-(pteridinyl)methyl)-methylamino]-benzoic acid + L-glutamate
-
-
-
-
?
methotrexate + H2O
4-[(2,4-diamino-6-(pteridinyl)methyl)-methylamino]-benzoic acid + L-glutamate
-
related toxicity, overview, rapid inactivation of methotrexate by CPDG2, the product DAMPA is eliminated more raoidly than methotrexate
i.e. DAMPA
-
?
additional information
?
-
-
rapid hydrolysis occurs in linkages of the form RCO-Glu and RCO-Gln, whereas those of the form RCO-D-Glu (D-Glu-D-Glu and Z-D-Glu) are not attacked
-
-
?
additional information
?
-
-
no hydrolysis of glutamate or glutamine lacking a free carboxyl alpha-carboxyl
-
-
?
additional information
?
-
-
effects of carboxypeptidase G2 administration to patients with methotrexate-induce renal failure, overview
-
-
?
additional information
?
-
-
enzyme is used in enzyme prodrug therapy to treat human breast cancer, the enzyme catalyzes the activation of the di- and trifluorinated prodrugs, overview
-
-
?
additional information
?
-
-
half-lives of prodrugs and drugs, overview
-
-
?
additional information
?
-
-
specifically cleaves terminal glutamic acid amines
-
-
?
additional information
?
-
-
CPG2 cleaves folic acid to pteroic acid and activates a range of bifunctional alkylating agent and antibiotic prodrugs
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(4-[bis(2-bromoethyl)amino]-3,5-difluorobenzoyl)-L-glutamic acid + H2O
4-[bis(2-bromoethyl)amino]-3,5-difluorobenzoic acid + L-glutamate
-
a difluorinated prodrug
-
-
?
(4-[bis(2-iodoethyl)amino]-3,5-difluorobenzoyl)-L-glutamic acid + H2O
4-[bis(2-iodoethyl)amino]-3,5-difluorobenzoic acid + L-glutamate
-
a difluorinated prodrug
-
-
?
diethylstilbestrol-glutamate + H2O
diethylstilbestrol + L-glutamate
-
the water soluble substrate can be used as a prodrug that is hydrolyzed to yield the active drug in an antibody directed enzyme prodrug therapy
-
-
?
methotrexate + H2O
4-[(2,4-diamino-6-(pteridinyl)methyl)-methylamino]-benzoic acid + L-glutamate
-
related toxicity, overview, rapid inactivation of methotrexate by CPDG2, the product DAMPA is eliminated more raoidly than methotrexate
i.e. DAMPA
-
?
additional information
?
-
-
effects of carboxypeptidase G2 administration to patients with methotrexate-induce renal failure, overview
-
-
?
additional information
?
-
-
enzyme is used in enzyme prodrug therapy to treat human breast cancer, the enzyme catalyzes the activation of the di- and trifluorinated prodrugs, overview
-
-
?
additional information
?
-
-
specifically cleaves terminal glutamic acid amines
-
-
?
additional information
?
-
-
CPG2 cleaves folic acid to pteroic acid and activates a range of bifunctional alkylating agent and antibiotic prodrugs
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Zn2+
additional information
Zn2+
-
each subunit of the dimer constists of a larger catalytic domain containing two zinc ions at the active site and a separate smaller domain that forms the dimer interface
Zn2+
-
contains 4 atoms of zinc per enzyme molecule, which are required for full activity
additional information
Mg2+ inhibits the activity of the recombinant enzyme
additional information
-
Mg2+ inhibits the activity of the recombinant enzyme
additional information
Mn2+ inhibits the activity of the recombinant enzyme
additional information
-
Mn2+ inhibits the activity of the recombinant enzyme
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
(4-[bis(2-chloroethyl)amino]-2,3,5,6-tetrafluorobenzoyl)-L-glutamic acid
-
prodrug, competitive inhibition
(4-[bis(2-iodoethyl)amino]-2,3,5,6-tetrafluorobenzoyl)-L-glutamic acid
-
prodrug, competitive inhibition
additional information
Mg2+ inhibits the activity of the recombinant enzyme
-
additional information
-
Mg2+ inhibits the activity of the recombinant enzyme
-
additional information
Mn2+ inhibits the activity of the recombinant enzyme
-
additional information
-
Mn2+ inhibits the activity of the recombinant enzyme
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0034
(2-chloroethyl)-(2-(methylsulfoyloxyethyl)amino)aminobenzyl-L-glutamic acid
-
pH 7.3, 37 °C
0.0011
(4-[bis(2-bromoethyl)amino]-2,3,5-trifluorobenzoyl)-L-glutamic acid
-
pH 7.3, 37 °C
0.0012
(4-[bis(2-bromoethyl)amino]-3,5-difluorobenzoyl)-L-glutamic acid
-
pH 7.3, 37 °C
0.0026
(4-[bis(2-chloroethyl)amino]-2,3,5-trifluorobenzoyl)-L-glutamic acid
-
pH 7.3, 37 °C
0.0017
(4-[bis(2-chloroethyl)amino]-3,5-difluorobenzoyl)-L-glutamic acid
-
pH 7.3, 37 °C
0.0028
(4-[bis(2-iodoethyl)amino]-2,3,5-trifluorobenzoyl)-L-glutamic acid
-
pH 7.3, 37 °C
0.0017
(4-[bis(2-iodoethyl)amino]-3,5-difluorobenzoyl)-L-glutamic acid
-
pH 7.3, 37 °C
0.0013
di-tert-butyl 4-[[2'-[N,N-bis(2''-chloroethyl)amino]phenyl]carbamoyloxymethyl]phenyloxycarbonyl-L-glutamic acid
-
-
0.00605
diallyl 4-[N-[4'-bis(2''-chloroethyl)aminophenyl]-N-methylcarbamoyloxymethyl]phenylcarbamoyl-L-glutamic acid
-
-
0.00055
diallyl 4-[[2'-[N,N-bis(2''-chloroethyl)amino]phenyl]carbamoyloxymethyl]phenylcarbamoyl-L-glutamic acid
-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
583
(2-chloroethyl)-(2-(methylsulfoyloxyethyl)amino)aminobenzyl-L-glutamic acid
-
pH 7.3, 37 °C
825
(4-[bis(2-bromoethyl)amino]-2,3,5-trifluorobenzoyl)-L-glutamic acid
-
pH 7.3, 37 °C
827
(4-[bis(2-bromoethyl)amino]-3,5-difluorobenzoyl)-L-glutamic acid
-
pH 7.3, 37 °C
1150
(4-[bis(2-chloroethyl)amino]-2,3,5-trifluorobenzoyl)-L-glutamic acid
-
pH 7.3, 37 °C
732
(4-[bis(2-chloroethyl)amino]-3,5-difluorobenzoyl)-L-glutamic acid
-
pH 7.3, 37 °C
220
(4-[bis(2-iodoethyl)amino]-2,3,5-trifluorobenzoyl)-L-glutamic acid
-
pH 7.3, 37 °C
1150
(4-[bis(2-iodoethyl)amino]-3,5-difluorobenzoyl)-L-glutamic acid
-
pH 7.3, 37 °C
5.5
diallyl 4-[N-[4'-bis(2''-chloroethyl)aminophenyl]-N-methylcarbamoyloxymethyl]phenylcarbamoyl-L-glutamic acid
-
-
2.9
diallyl 4-[[2'-[N,N-bis(2''-chloroethyl)amino]phenyl]carbamoyloxymethyl]phenylcarbamoyl-L-glutamic acid
-
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
83000
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
activity can increase up to 30% if the enzyme at all stages of purification is stored at -20°C
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
as His-tagged protein in a single step by Ni2+ charged column chromatography
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
overexpression of a codon-optimized gene in Escherichia coli, vector pET28a, the enzyme is expressed to about 60% of the total host protein and the purification of the recombinant His-tagged protein could be achieved in a single step by Ni2+ charged column chromatography
RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
Refolding is initiated by rapid 25fold dilution of the denatured protein into refolding buffer (100 mM Tris-HCl, pH 8.5, containing 0.1 mM EDTA and 0.5 M arginine)
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
medicine
glutamate chemical exchange saturation transfer magnetic resonance imaging GluCEST-MRI is an attractive method for metabolic imaging of glutamate. The change in glutamate concentration measured with GluCEST MRI could provide a noninvasive biomarker of carboxypeptidase G2 activity, in addition to acting as a surrogate for prodrug activation and the concentration of activated drug in the tumor
drug development
-
enzyme is used in enzyme prodrug therapy to treat human breast cancer, the enzyme catalyzes the activation of the di- and trifluorinated prodrugs
medicine
medicine
-
enzyme is used in enzyme prodrug therapy to treat human breast cancer, the enzyme catalyzes the activation of the di- and trifluorinated prodrugs
medicine
-
CPG2 possesses several properties that make it suitable for suicide gene therapy
medicine
-
carboxypeptidase G2 is a bacterial enzyme that is currently employed in a range of targeted cancer chemotherapy strategies such as gene-directed enzyme prodrug therapy (GDEPT). These therapeutic strategies would benefit from robust imaging strategies that afford high spatial and temporal resolution images of the biodistribution of carboxypeptidase G2 activity. Employing dynamic nuclear polarization (DNP) and natural abundance 13C magnetic resonance spectroscopy (MRS), the carboxypeptidase G2-mediated conversion of a novel hyperpolarized reporter probe 3,5-difluorobenzoyl-L-glutamic acid to 3,5-difluorobenzoic acid and L-glutamic acid is observed in vitro. The potential for translation in vivo will primarily depend on the 13C relaxation times and this appears to be an important limiting factor in the case of 3,5-difluorobenzoyl-L-glutamic acid
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Goldman, P.; Levy, C.C.
Carboxypeptidase G: purification and properties
Proc. Natl. Acad. Sci. USA
58
1299-1306
1967
Pseudomonas sp.
Sherwood, R.F.; Melton, R.G.; Alwan, S.M.; Hughes, P.
Purification and properties of carboxypeptidase G2 from Pseudomonas sp. strain RS-16. Use of a novel triazine dye affinity method
Eur. J. Biochem.
148
447-453
1985
Pseudomonas sp.
Lloyd, L.F.; Collyer, C.A.; Sherwood, R.F.
Crystallization and preliminary crystallographic analysis of carboxypeptidase G2 from Pseudomonas sp. strain RS-16
J. Mol. Biol.
220
17-18
1991
Pseudomonas sp.
Rowsell, S.; Pauptit, R.A.; Tucker, A.D.; Melton, R.G.; Blow, D.M.; Brick, P.
Crystal structure of carboxypeptidase G2, a bacterial enzyme with applications in cancer therapy
Structure
5
337-347
1997
Pseudomonas sp.
Niculescu-Duvaz, D.; Niculescu-Duvaz, I.; Friedlos, F.; Martin, J.; Lehouritis, P.; Marais, R.; Springer, C.J.
Self-immolative nitrogen mustards prodrugs cleavable by carboxypeptidase G2 (CPG2) showing large cytotoxicity differentials in GDEPT
J. Med. Chem.
46
1690-1705
2003
Pseudomonas sp.
Buchen, S.; Ngampolo, D.; Melton, R.G.; Hasan, C.; Zoubek, A.; Henze, G.; Bode, U.; Fleischhack, G.
Carboxypeptidase G2 rescue in patients with methotrexate intoxication and renal failure
Br. J. Cancer
92
480-487
2005
Pseudomonas sp.
Davies, L.C.; Friedlos, F.; Hedley, D.; Martin, J.; Ogilvie, L.M.; Scanlon, I.J.; Springer, C.J.
Novel fluorinated prodrugs for activation by carboxypeptidase G2 showing good in vivo antitumor activity in gene-directed enzyme prodrug therapy
J. Med. Chem.
48
5321-5328
2005
Pseudomonas sp., Pseudomonas sp. RS16
Pedone, E.; Searle, F.; Brocchini, S.
Diethylstilbestrol glutamate as a potential substrate for ADEPT
J. Drug Target.
14
437-443
2006
Pseudomonas sp.
Friedlos, F.; Lehouritis, P.; Ogilvie, L.; Hedley, D.; Davies, L.; Bermudes, D.; King, I.; Martin, J.; Marais, R.; Springer, C.J.
Attenuated Salmonella targets prodrug activating enzyme carboxypeptidase G2 to mouse melanoma and human breast and colon carcinomas for effective suicide gene therapy
Clin. Cancer Res.
14
4259-4266
2008
Pseudomonas sp.
Jamin, Y.; Gabellieri, C.; Smyth, L.; Reynolds, S.; Robinson, S.P.; Springer, C.J.; Leach, M.O.; Payne, G.S.; Eykyn, T.R.
Hyperpolarized (13)C magnetic resonance detection of carboxypeptidase G2 activity
Magn. Reson. Med.
62
1300-1304
2009
Pseudomonas sp.
Goda, S.K.; Rashidi, F.A.; Fakharo, A.A.; Al-Obaidli, A.
Functional overexpression and purification of a codon optimized synthetic glucarpidase (carboxypeptidase G2) in Escherichia coli
Protein J.
28
435-442
2009
Pseudomonas sp. (P06621), Pseudomonas sp.
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