BRENDA - Enzyme Database show
show all sequences of 3.1.1.84

Biochemical characterization and structural analysis of a highly proficient cocaine esterase

Turner, J.M.; Larsen, N.A.; Basran, A.; Barbas, C.F., 3rd; Bruce, N.C.; Wilson, I.A.; Lerner, R.A.; Biochemistry 41, 12297-12307 (2002)

Data extracted from this reference:

Application
Application
Commentary
Organism
medicine
the high catalytic proficiency, lack of observable product inhibition, and ability to hydrolyze both cocaine and cocaethylene make cocE an attractive candidate for rapid cocaine detoxification in an emergency setting
Rhodococcus sp.
Cloned(Commentary)
Commentary
Organism
-
Rhodococcus sp.
Crystallization (Commentary)
Crystallization
Organism
crystal structures of the S117A and Y44F mutants of cocE
Rhodococcus sp.
Engineering
Amino acid exchange
Commentary
Organism
D259N
mutation results in more than 1500fold decrease in kcat
Rhodococcus sp.
F261A
mutant catalyzed the hydrolysis of cocaine with a 29fold lower kcat and 15fold higher KM
Rhodococcus sp.
F408A
mutant has 8fold increased KM and more than 100fold decrease in kcat
Rhodococcus sp.
H287A
mutation results in more than 1500fold decrease in kcat
Rhodococcus sp.
L407A
mutant has 2fold increased KM and more than 100fold decrease in kcat
Rhodococcus sp.
L407A/F408A
attempts to express the L407A/F408A double mutant do not result in any soluble protein
Rhodococcus sp.
Q55E
the mutation within the active site of cocE results in a 2fold improvement in KM, but a 14fold loss of kcat
Rhodococcus sp.
S117A
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
Rhodococcus sp.
W151A
mutant catalyzed the hydrolysis of cocaine with a 78fold lower kcat and 80fold higher KM
Rhodococcus sp.
W166A
mutant has a 29fold lower kcat, and a 6fold increased KM
Rhodococcus sp.
Y44F
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
Rhodococcus sp.
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.00027
-
cocaine
pH 7.4, mutant enzyme Q55E
Rhodococcus sp.
0.00064
-
cocaine
pH 7.4, wild-type enzyme
Rhodococcus sp.
0.00075
-
cocaine
pH 7.4, mutant enzyme Q55A
Rhodococcus sp.
0.0012
-
cocaine
pH 7.4, mutant enzyme L407A
Rhodococcus sp.
0.0016
-
cocaethylene
pH 7.4, wild-type enzyme
Rhodococcus sp.
0.0036
-
cocaine
pH 7.4, mutant enzyme W166A
Rhodococcus sp.
0.0051
-
cocaine
pH 7.4, mutant enzyme F408A
Rhodococcus sp.
0.0096
-
cocaine
pH 7.4, mutant enzyme F261A
Rhodococcus sp.
0.046
-
cocaine
pH 7.4, mutant enzyme S117C
Rhodococcus sp.
0.051
-
cocaine
pH 7.4, mutant enzyme W151A
Rhodococcus sp.
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
cocaine + H2O
Rhodococcus sp.
-
ecgonine methyl ester + benzoate
-
-
?
Organic Solvent Stability
Organic Solvent
Commentary
Organism
urea
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
Rhodococcus sp.
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Rhodococcus sp.
Q9L9D7
-
-
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
cocaethylene + H2O
cocaethylene is a more potent cocaine metabolite, observed in patients who concurrently abuse cocaine and alcohol
650108
Rhodococcus sp.
?
-
-
-
?
cocaine + H2O
-
650108
Rhodococcus sp.
ecgonine methyl ester + benzoate
-
-
-
?
cocaine + H2O
the bacterial cocaine esterase, cocE, hydrolyzes cocaine faster than any other reported cocaine esterase
650108
Rhodococcus sp.
ecgonine methyl ester + benzoate
-
-
-
?
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.046
-
cocaine
pH 7.4, mutant enzyme S117C
Rhodococcus sp.
0.057
-
cocaine
pH 7.4, mutant enzyme F408A
Rhodococcus sp.
0.067
-
cocaine
pH 7.4, mutant enzyme L407A
Rhodococcus sp.
0.1
-
cocaine
pH 7.4, mutant enzyme W151A
Rhodococcus sp.
0.27
-
cocaine
pH 7.4, mutant enzyme F261A; pH 7.4, mutant enzyme W166A
Rhodococcus sp.
0.55
-
cocaine
pH 7.4, mutant enzyme Q55E
Rhodococcus sp.
1.7
-
cocaine
pH 7.4, mutant enzyme Q55A
Rhodococcus sp.
7.8
-
cocaine
pH 7.4, wild-type enzyme
Rhodococcus sp.
9.4
-
cocaethylene
pH 7.4, wild-type enzyme
Rhodococcus sp.
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
9
-
-
Rhodococcus sp.
pH Range
pH Minimum
pH Maximum
Commentary
Organism
7.8
10.5
pH 7.8: about 50% of maximal activity, pH 10.0: about 50% of maximal activity
Rhodococcus sp.
Application (protein specific)
Application
Commentary
Organism
medicine
the high catalytic proficiency, lack of observable product inhibition, and ability to hydrolyze both cocaine and cocaethylene make cocE an attractive candidate for rapid cocaine detoxification in an emergency setting
Rhodococcus sp.
Cloned(Commentary) (protein specific)
Commentary
Organism
-
Rhodococcus sp.
Crystallization (Commentary) (protein specific)
Crystallization
Organism
crystal structures of the S117A and Y44F mutants of cocE
Rhodococcus sp.
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
D259N
mutation results in more than 1500fold decrease in kcat
Rhodococcus sp.
F261A
mutant catalyzed the hydrolysis of cocaine with a 29fold lower kcat and 15fold higher KM
Rhodococcus sp.
F408A
mutant has 8fold increased KM and more than 100fold decrease in kcat
Rhodococcus sp.
H287A
mutation results in more than 1500fold decrease in kcat
Rhodococcus sp.
L407A
mutant has 2fold increased KM and more than 100fold decrease in kcat
Rhodococcus sp.
L407A/F408A
attempts to express the L407A/F408A double mutant do not result in any soluble protein
Rhodococcus sp.
Q55E
the mutation within the active site of cocE results in a 2fold improvement in KM, but a 14fold loss of kcat
Rhodococcus sp.
S117A
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
Rhodococcus sp.
W151A
mutant catalyzed the hydrolysis of cocaine with a 78fold lower kcat and 80fold higher KM
Rhodococcus sp.
W166A
mutant has a 29fold lower kcat, and a 6fold increased KM
Rhodococcus sp.
Y44F
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
Rhodococcus sp.
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.00027
-
cocaine
pH 7.4, mutant enzyme Q55E
Rhodococcus sp.
0.00064
-
cocaine
pH 7.4, wild-type enzyme
Rhodococcus sp.
0.00075
-
cocaine
pH 7.4, mutant enzyme Q55A
Rhodococcus sp.
0.0012
-
cocaine
pH 7.4, mutant enzyme L407A
Rhodococcus sp.
0.0016
-
cocaethylene
pH 7.4, wild-type enzyme
Rhodococcus sp.
0.0036
-
cocaine
pH 7.4, mutant enzyme W166A
Rhodococcus sp.
0.0051
-
cocaine
pH 7.4, mutant enzyme F408A
Rhodococcus sp.
0.0096
-
cocaine
pH 7.4, mutant enzyme F261A
Rhodococcus sp.
0.046
-
cocaine
pH 7.4, mutant enzyme S117C
Rhodococcus sp.
0.051
-
cocaine
pH 7.4, mutant enzyme W151A
Rhodococcus sp.
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
cocaine + H2O
Rhodococcus sp.
-
ecgonine methyl ester + benzoate
-
-
?
Organic Solvent Stability (protein specific)
Organic Solvent
Commentary
Organism
urea
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
Rhodococcus sp.
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
cocaethylene + H2O
cocaethylene is a more potent cocaine metabolite, observed in patients who concurrently abuse cocaine and alcohol
650108
Rhodococcus sp.
?
-
-
-
?
cocaine + H2O
-
650108
Rhodococcus sp.
ecgonine methyl ester + benzoate
-
-
-
?
cocaine + H2O
the bacterial cocaine esterase, cocE, hydrolyzes cocaine faster than any other reported cocaine esterase
650108
Rhodococcus sp.
ecgonine methyl ester + benzoate
-
-
-
?
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.046
-
cocaine
pH 7.4, mutant enzyme S117C
Rhodococcus sp.
0.057
-
cocaine
pH 7.4, mutant enzyme F408A
Rhodococcus sp.
0.067
-
cocaine
pH 7.4, mutant enzyme L407A
Rhodococcus sp.
0.1
-
cocaine
pH 7.4, mutant enzyme W151A
Rhodococcus sp.
0.27
-
cocaine
pH 7.4, mutant enzyme F261A; pH 7.4, mutant enzyme W166A
Rhodococcus sp.
0.55
-
cocaine
pH 7.4, mutant enzyme Q55E
Rhodococcus sp.
1.7
-
cocaine
pH 7.4, mutant enzyme Q55A
Rhodococcus sp.
7.8
-
cocaine
pH 7.4, wild-type enzyme
Rhodococcus sp.
9.4
-
cocaethylene
pH 7.4, wild-type enzyme
Rhodococcus sp.
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
9
-
-
Rhodococcus sp.
pH Range (protein specific)
pH Minimum
pH Maximum
Commentary
Organism
7.8
10.5
pH 7.8: about 50% of maximal activity, pH 10.0: about 50% of maximal activity
Rhodococcus sp.
Other publictions for EC 3.1.1.84
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
750291
Kim
Kinetic characterization of c ...
Homo sapiens
Chem. Biol. Interact.
293
107-114
2018
-
-
1
-
-
-
1
3
-
-
-
3
-
1
-
-
-
-
-
-
-
-
5
-
1
-
-
3
1
-
-
-
1
-
-
-
-
1
-
-
-
-
-
1
1
3
-
-
-
3
-
-
-
-
-
-
-
-
5
-
1
-
-
3
1
-
-
-
-
1
1
-
3
3
728856
Fang
Rational design, preparation, ...
Rhodococcus sp., Rhodococcus sp. MB1 Bresler
ACS Chem. Biol.
9
1764-1772
2014
-
-
1
1
3
-
-
3
-
-
-
2
-
3
-
-
1
-
-
-
-
-
2
1
-
-
-
3
-
-
-
-
-
-
-
-
-
1
-
1
3
-
-
-
-
3
-
-
-
2
-
-
-
1
-
-
-
-
2
1
-
-
-
3
-
-
-
-
-
-
-
-
3
3
730250
Brim
The fate of bacterial cocaine ...
Rhodococcus sp.
J. Pharmacol. Exp. Ther.
340
83-95
2012
-
-
1
-
-
-
-
-
-
-
1
1
-
4
-
-
1
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
1
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
730421
Lee
Cell permeable cocaine esteras ...
Rhodococcus sp., Rhodococcus sp. MB1
Mol. Pharm.
9
1361-1373
2012
-
-
1
-
5
-
-
6
-
-
1
2
-
6
-
-
1
-
-
-
-
-
2
1
-
-
1
6
-
-
-
-
-
-
-
-
-
1
-
-
5
-
-
-
-
6
-
-
1
2
-
-
-
1
-
-
-
-
2
1
-
-
1
6
-
-
-
-
-
-
-
-
6
6
716278
Xue
Design, preparation, and chara ...
Homo sapiens
Mol. Pharmacol.
79
290-297
2011
-
-
1
-
3
-
-
4
-
-
-
-
-
1
-
-
1
-
-
1
-
-
1
-
-
-
-
4
-
-
-
-
-
-
-
-
-
1
-
-
3
-
-
-
-
4
-
-
-
-
-
-
-
1
-
1
-
-
1
-
-
-
-
4
-
-
-
-
-
-
-
-
4
4
716281
Narasimhan
Subunit stabilization and pegy ...
Rhodococcus sp.
Mol. Pharmacol.
80
1056-1065
2011
-
-
1
1
3
-
-
4
-
-
1
-
-
3
-
-
1
-
-
-
-
-
1
1
-
-
1
4
-
-
-
-
-
-
-
-
-
1
-
1
3
-
-
-
-
4
-
-
1
-
-
-
-
1
-
-
-
-
1
1
-
-
1
4
-
-
-
-
-
-
-
-
4
4
714226
Zheng
Design of high-activity mutant ...
Homo sapiens
Biochemistry
49
9113-9119
2010
-
-
1
1
10
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
1
10
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
9
9
714702
Yang
Characterization of a high-act ...
Homo sapiens
Chem. Biol. Interact.
187
148-152
2010
-
-
-
-
1
-
-
2
-
-
-
-
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
2
-
-
-
-
-
-
-
-
2
2
716276
Brim
A thermally stable form of bac ...
Rhodococcus sp., Rhodococcus sp. MB1
Mol. Pharmacol.
77
593-600
2010
-
1
-
1
1
-
-
4
-
-
-
-
-
7
-
-
-
-
-
-
-
-
2
-
-
-
1
-
-
-
-
-
-
-
-
-
1
-
-
1
1
-
-
-
-
4
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
716824
Narasimhan
Structural analysis of thermos ...
Rhodococcus sp., Rhodococcus sp. MB1
Protein Eng.
23
537-547
2010
-
1
1
-
5
-
-
4
-
-
-
-
-
6
-
-
1
-
-
-
-
-
2
-
-
-
1
5
-
-
-
-
-
-
-
-
1
1
-
-
5
-
-
-
-
4
-
-
-
-
-
-
-
1
-
-
-
-
2
-
-
-
1
5
-
-
-
-
-
-
-
-
5
5
692028
Ko
Effects of cocaine esterase fo ...
Rhodococcus sp.
Drug Alcohol Depend.
101
202-209
2009
-
1
-
-
-
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
694267
Gao
Thermostable variants of cocai ...
Rhodococcus sp.
Mol. Pharmacol.
75
318-323
2009
-
1
-
-
4
-
-
3
-
-
-
1
-
1
-
-
1
-
-
-
-
-
2
-
1
-
1
4
1
-
-
-
-
-
-
-
1
-
-
-
4
-
-
-
-
3
-
-
-
1
-
-
-
1
-
-
-
-
2
-
1
-
1
4
1
-
-
-
-
-
-
-
-
-
699760
Collins
Zhan. C.G.; Woods. J.H.: Cocai ...
Rhodococcus sp.
J. Pharmacol. Exp. Ther.
331
445-455
2009
-
1
-
-
1
-
-
-
-
-
-
-
-
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
698483
Liu
Fundamental reaction mechanism ...
Rhodococcus sp.
J. Am. Chem. Soc.
131
11964-11975
2003
-
-
-
-
-
-
-
-
-
-
-
1
-
1
-
-
-
1
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
650108
Turner
Biochemical characterization a ...
Rhodococcus sp.
Biochemistry
41
12297-12307
2002
-
1
1
1
11
-
-
10
-
-
-
1
1
2
-
-
-
-
-
-
-
-
3
-
-
-
-
9
1
1
-
-
-
-
-
-
1
1
-
1
11
-
-
-
-
10
-
-
-
1
1
-
-
-
-
-
-
-
3
-
-
-
-
9
1
1
-
-
-
-
-
-
-
-
653322
Larsen
Crystal structure of a bacteri ...
Rhodococcus sp.
Nat. Struct. Biol.
9
17-21
2002
-
1
-
1
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
695729
Bresler
Gene cloning and nucleotide se ...
Rhodococcus sp., Rhodococcus sp. MB1
Appl. Environ. Microbiol.
66
904-908
2000
-
-
1
-
-
-
-
1
-
-
2
2
-
11
-
-
1
-
-
1
1
-
6
1
1
-
-
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
1
-
-
2
2
-
-
-
1
-
1
1
-
6
1
1
-
-
-
1
-
-
-
-
-
-
-
-
-
171019
Pindel
Purification and cloning of a ...
Homo sapiens
J. Biol. Chem.
272
14769-14775
1997
-
-
1
-
-
-
1
4
-
-
2
1
-
1
-
1
1
-
-
1
-
-
5
1
-
-
-
-
-
-
-
-
1
1
-
-
-
1
-
-
-
-
-
1
1
4
-
-
2
1
-
-
1
1
-
1
-
-
5
1
-
-
-
-
-
-
-
1
-
-
-
-
-
-
698540
Britt
Identification of a cocaine es ...
Stenotrophomonas maltophilia, Stenotrophomonas maltophilia MB11L
J. Bacteriol.
174
2087-2094
1992
-
-
-
-
-
-
2
3
-
-
3
2
-
4
-
-
1
-
-
-
1
-
10
2
1
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
3
-
-
3
2
-
-
-
1
-
-
1
-
10
2
1
-
-
-
1
-
-
-
-
-
-
-
-
-