Information on EC 3.1.1.84 - cocaine esterase

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

EC NUMBER
COMMENTARY hide
3.1.1.84
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RECOMMENDED NAME
GeneOntology No.
cocaine esterase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
cocaine + H2O = ecgonine methyl ester + benzoate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Tropane, piperidine and pyridine alkaloid biosynthesis
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-
Metabolic pathways
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Biosynthesis of secondary metabolites
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SYSTEMATIC NAME
IUBMB Comments
cocaine benzoylhydrolase
Rhodococcus sp. strain MB1 and Pseudomonas maltophilia strain MB11L can utilize cocaine as sole source of carbon and energy [2,3].
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(-)-cocaine + H2O
benzoic acid + methyl (1R,2R,3S,5S)-3-hydroxy-8-methyl-8-azabicyclo[3.2.1]-octane-2-carboxylate
show the reaction diagram
-
-
i.e. ecgonine methyl ester
-
?
(-)-cocaine + H2O
benzoic acid + methyl-(1R,2R,3S,5S)-3-hydroxy-8-methyl-8-azabicyclo[3.2.1]-octane-2-carboxylate
show the reaction diagram
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-
i.e. ecgonine methyl ester
-
?
(-)-cocaine + H2O
ecgonine methyl ester + benzoate
show the reaction diagram
4-methylumbelliferyl acetate + H2O
4-methylumbelliferone + acetate
show the reaction diagram
hCE-2 has higher catalytic efficiency for hydrolysis than hCE-1
-
-
?
6-monoacetylmorphine + H2O
morphine + acetate
show the reaction diagram
hCE-2 has higher catalytic efficiency for hydrolysis than hCE-1
-
-
?
cocaethylene + H2O
?
show the reaction diagram
cocaethylene is a more potent cocaine metabolite, observed in patients who concurrently abuse cocaine and alcohol
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-
?
cocaine + H2O
ecgonine methyl ester + benzoate
show the reaction diagram
ethyl 2-hydroxybenzoate + H2O
ethanol + 2-hydroxybenzoate
show the reaction diagram
ethyl benzoate + H2O
ethanol + benzoate
show the reaction diagram
heroin + H2O
6-monoacetylmorphine + acetate
show the reaction diagram
hCE-2 has higher catalytic efficiency for hydrolysis than hCE-1
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-
?
additional information
?
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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
(-)-cocaine + H2O
ecgonine methyl ester + benzoate
show the reaction diagram
-
-
-
-
?
cocaine + H2O
ecgonine methyl ester + benzoate
show the reaction diagram
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
eserine
hCE-2 shows greater inhibition by eserine thann hCE-1
phenylmethylsulfonyl fluoride
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1 mM, complete inhibition
additional information
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no inhibition by 1 mM eserine or 1 mM p-hydroxymercuribenzoate. Cocaine esterase shows product inhibition with neither benzoate nor ecgonine methyl ester
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0011 - 0.0045
(-)-cocaine
0.15
4-yethylumbelliferyl acetate
pH 7.4, 37C
0.13
6-monoacetylmorphine
pH 7.4, 37C
0.0016
cocaethylene
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pH 7.4, wild-type enzyme
0.00027 - 1.33
cocaine
1.75
ethyl 2-hydroxybenzoate
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pH 7.0, 30C
1.89
ethyl benzoate
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pH 7.0, 30C
6.8
heroin
pH 7.4, 37C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.002 - 73.83
(-)-cocaine
9.4
cocaethylene
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pH 7.4, wild-type enzyme
0.046 - 2502
cocaine
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
15.17 - 30000
(-)-cocaine
702 - 38330
cocaine
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0001
eserine
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4
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assay at
7.5
-
assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.8 - 10.5
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pH 7.8: about 50% of maximal activity, pH 10.0: about 50% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
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assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.9
isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Rhodococcus sp. (strain MB1 Bresler)
Rhodococcus sp. (strain MB1 Bresler)
Rhodococcus sp. (strain MB1 Bresler)
Rhodococcus sp. (strain MB1 Bresler)
Rhodococcus sp. (strain MB1 Bresler)
Rhodococcus sp. (strain MB1 Bresler)
Rhodococcus sp. (strain MB1 Bresler)
Rhodococcus sp. (strain MB1 Bresler)
Rhodococcus sp. (strain MB1 Bresler)
Rhodococcus sp. (strain MB1 Bresler)
Rhodococcus sp. (strain MB1 Bresler)
Rhodococcus sp. (strain MB1 Bresler)
Rhodococcus sp. (strain MB1 Bresler)
Rhodococcus sp. (strain MB1 Bresler)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60000
1 * 60000, SDS-PAGE
62128
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1 * 62128, calculated from sequence
70000
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x * 70000, SDS-PAGE
80000
gel filtration
110000
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in the presence of the solubilizing agent cholate, gel filtration
127000
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129000
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1 * 129000, SDS-PAGE
410000
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in the absence of cholate, cocaine esterase has a native MW of 410000 and probably existed as a tetramer, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
monomer
tetramer
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
high mannose type
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structures of the S117A and Y44F mutants of cocE
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hanging drop vapor diffusion method, using 0.1 M phosphate-citrate (pH 4.2), 1.6 M sodium dihydrogen phosphate, and 0.4 M dipotassium hydrogen phosphate
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hanging drop vapor diffusion method, using 20% (w/v) PEG 3350, 100 mM 2-(N-morpholino)-ethane sulfonic acid, pH 6.0, and 1 M NaCl
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mutant enzyme L169K/G173Q, hanging drop vapor diffusion method, using 1.7 M ammonium sulfate, 10 mM Tris-HCl, pH 7.3, and 25 mM NaCl
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te crystal structure of cocE, solved by multiple anomalous dispersion methods, reveals that cocE is a serine esterase composed of three domains: (1.) a canonical alpha/beta hydrolase fold (2.) an alpha-helical domain that caps the active site and (3.) a jelly-roll-like beta-domain that interacts extensively with the other two domains. The active site is identified within the interface of all three domains by analysis of the crystal structures of transition state analog adduct and product complexes, which are refined at 1.58 A and 1.63 A resolution, respectively
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
urea
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urea denaturation studies of cocE by fluorescence and circular dichroism show two unfolding transitions (0.5-0.6 M and 3.2-3.7 M urea), with the first transition likely representing pertubation of the active site
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
HisPur cobalt resin column chromatography
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Q-Sepharose column chromatography and phenyl Sepharose column chromatography
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QFF resin anion-exchange chromatography and Hypatite C column chromatography
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recombinant enzyme
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Talon metal affinity column chromatography and Q-Sepharose column chromatography
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Talon metal chelate affinity column chromatography and Q-Sepharose column chromatography
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Talon metal chelate column chromatography and Q-Sepharose column chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli B834 (DE3) cells
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expressed in Escherichia coli BL-21 Gold (DE3) cells
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expressed in Escherichia coli BL21 Star (DE3) cells
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expressed in Escherichia coli BL21(DE3) cells
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expressed in HEK-293T/17 cells
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the cocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed in Escherichia coli. sequence comparison suggests that cocE encodes a serine esterase
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wild type and mutant enzymes are expressed in HEK-293T/17 cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A199S/F227A/S287G/A328W/E441D
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the mutant shows 1730fold improved (-)-cocaine-hydrolyzing activity compared to the wild type enzyme
A199S/F227A/S287G/A328W/Y332G
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the mutant has a 2020fold improved catalytic efficiency against (-)-cocaine compared to the wild type enzyme
A199S/F227A/S287G/A328W/Y332G/E441D
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the mutant shows 1390fold improved (-)-cocaine-hydrolyzing activity compared to the wild type enzyme
A199S/F227A/S287G/A328W/Y332G/F329V
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the mutant has a 121fold improved catalytic efficiency against (-)-cocaine compared to the wild type enzyme
A199S/F227I/S287G/A328W/Y332G
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the mutant has a 1170fold improved catalytic efficiency against (-)-cocaine compared to the wild type enzyme
A199S/F227L/S287G/A328W/Y332G
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the mutant has a 1130fold improved catalytic efficiency against (-)-cocaine compared to the wild type enzyme
A199S/F227V/S287G/A328W/Y332G
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the mutant has a 1490fold improved catalytic efficiency against (-)-cocaine compared to the wild type enzyme
A199S/S287G/A328W/Y332G
A199S/S287G/A328W/Y332G/L286I
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the mutant has a 242fold improved catalytic efficiency against (-)-cocaine compared to the wild type enzyme
A328W/Y332A
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the mutant has a 9.4fold improved catalytic efficiency against (-)-cocaine compared to the wild type enzyme
A328W/Y332G
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the mutant has a 15fold improved catalytic efficiency against (-)-cocaine compared to the wild type enzyme
F227A/S287G/A328W/Y332M
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the mutant has a 34fold improved catalytic efficiency against (-)-cocaine compared to the wild type enzyme
D259N
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mutation results in more than 1500fold decrease in kcat
F261A
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mutant catalyzed the hydrolysis of cocaine with a 29fold lower kcat and 15fold higher KM
F408A
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mutant has 8fold increased KM and more than 100fold decrease in kcat
G173Q/L169K
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the mutant has a half-life of 370 min and 2.9 days at 37C
G4C/S10C
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the mutant shows about 4fold reduced catalytic efficiency compared to the wild type enzyme. The mutant retains almost all activity after 7 days of 37C treatment
H287A
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mutation results in more than 1500fold decrease in kcat
L169K
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the mutation significantly increases the stability of cocaine esterase over that of wild type enzyme (half-life at 37C is 570 min). The mutant exhibits about 8fold increase in Km for cocaine compared to the wild type enzyme
L169K/G173Q
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highly thermostable mutant with a half-life of 2.9 days at 37C
L407A
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mutant has 2fold increased KM and more than 100fold decrease in kcat
L407A/F408A
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attempts to express the L407A/F408A double mutant do not result in any soluble protein
LMWP-S-S-T172R/G173Q
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the mutant shows reduced catalytic efficiency compared to the wild type enzyme
LMWP-T172R/G173Q
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the mutant shows reduced catalytic efficiency compared to the wild type enzyme
Q55E
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the mutation within the active site of cocE results in a 2fold improvement in KM, but a 14fold loss of kcat
S117A
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mutation results in more than 1500fold decrease in kcat, crystal structures of the S117A and Y44F mutants of cocE. The first urea unfolding transition in the S117A mutant is shifted from 0.5 to 1.3 M urea compared to the wild-type, while the second transition, although broader, has a similar transition point
T172R/G173Q
T172R/G173Q -LMWP
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the mutant shows reduced catalytic efficiency compared to the wild type enzyme
T172R/G173Q -YGRKKRRQRRR
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the mutant shows reduced catalytic efficiency compared to the wild type enzyme
T172R/G173Q /G4C/S10C
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the mutant shows improved catalytic efficiency against cocaine by about 20%
T172R/G173Q/L169K
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the mutant shows poor enzyme kinetics and does not display enhanced stabilization
T172R/G173Q/L196C/I301C
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the mutant has not only considerably extended the in vitro half-life at 37C to more than 100 days, but also significantly improved catalytic efficiency against cocaine by about 150%
W151A
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mutant catalyzed the hydrolysis of cocaine with a 78fold lower kcat and 80fold higher KM
W166A
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mutant has a 29fold lower kcat, and a 6fold increased KM
Y44F
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mutation results in more than 1500fold decrease in kcat, crystal structures of the S117A and Y44F mutants of cocE. The urea unfolding curve of the Y44F mutant is very similar to the wild-type, and has almost identical transition points
YGRKKRRQRRR-T172R/G173Q
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the mutant still maintains 52% of its cocaine-hydrolyzing efficiency even after incubation at 37C for 24 h
G173Q
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the mutant does not have any deleterious effects on the catalytic efficiency
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L169K
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the mutation significantly increases the stability of cocaine esterase over that of wild type enzyme (half-life at 37C is 570 min). The mutant exhibits about 8fold increase in Km for cocaine compared to the wild type enzyme
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L169K/G173Q
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highly thermostable mutant with a half-life of 2.9 days at 37C
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LMWP-S-S-T172R/G173Q
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the mutant shows reduced catalytic efficiency compared to the wild type enzyme
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LMWP-T172R/G173Q
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the mutant shows reduced catalytic efficiency compared to the wild type enzyme
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T172R
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the mutants shows about wild type thermal stability and decreased catalytic efficiency for cocaine
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T172R/G173Q
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the mutation extends half-life at 37C up to 370 min (30fold improvement compared to the wild type stability) and leads to about 3fold decrease of catalytic efficiency for cocaine
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T172R/G173Q -LMWP
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the mutant shows reduced catalytic efficiency compared to the wild type enzyme
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T172R/G173Q -YGRKKRRQRRR
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the mutant shows reduced catalytic efficiency compared to the wild type enzyme
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T172R/G173Q/L169K
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the mutant shows poor enzyme kinetics and does not display enhanced stabilization
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YGRKKRRQRRR-T172R/G173Q
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the mutant still maintains 52% of its cocaine-hydrolyzing efficiency even after incubation at 37C for 24 h
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T172R/G173Q
Rhodococcus sp. MB1 Bresler
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the mutant has an improved in vitro half-life of about 6 h at 37C
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T172R/G173Q /G4C/S10C
Rhodococcus sp. MB1 Bresler
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the mutant shows improved catalytic efficiency against cocaine by about 20%
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T172R/G173Q/L196C/I301C
Rhodococcus sp. MB1 Bresler
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the mutant has not only considerably extended the in vitro half-life at 37C to more than 100 days, but also significantly improved catalytic efficiency against cocaine by about 150%
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additional information
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computational-experimental effort yields a CocE variant with a 30-fold increase in plasma half-life both in vitro and in vivo
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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