BRENDA - Enzyme Database show
show all sequences of 6.3.3.6

Inhibition and alternate substrate studies on the mechanism of carbapenam synthetase from Erwinia carotovora

Gerratana, B.; Stapon, A.; Townsend, C.A.; Biochemistry 42, 7836-7847 (2003)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
overexpression of the plasmid pET24a/carA transformed in Escherichia coli BL21(DE3) cells
Pectobacterium carotovorum
Inhibitors
Inhibitors
Commentary
Organism
Structure
(3S,5S)-carbapenam 3-carboxylate
-
Pectobacterium carotovorum
AMP
uncompetitive inhibition versus both substrates
Pectobacterium carotovorum
diphosphate
the competitive inhibition exhibited by diphosphate versus ATP allows the assignment of diphosphate as the last product released
Pectobacterium carotovorum
L-proline
the uncompetitive nature of the inhibition of the dead-end inhibitor, L-proline, versus ATP is consistent with a mechanism with ordered substrate binding where ATP binds first followed by (2S,5S)-carboxymethylproline
Pectobacterium carotovorum
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.11
-
ATP
pH 7.8, 22°C
Pectobacterium carotovorum
0.23
-
(2S,5S)-5-carboxymethylproline
pH 7.8, 22°C
Pectobacterium carotovorum
4.6
-
(2R,5R)-5-carboxymethylproline
pH 7.8, 22°C
Pectobacterium carotovorum
7
-
(2S,5R)-5-carboxymethylproline
pH 7.8, 22°C
Pectobacterium carotovorum
Molecular Weight [Da]
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
56000
-
2 * 56000, calculated from sequence
Pectobacterium carotovorum
119000
-
native PAGE
Pectobacterium carotovorum
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ATP + (2S,5S)-5-carboxymethylproline
Pectobacterium carotovorum
the enzyme is involved in the biosynthesis of the carbapenem beta-lactam antibiotic (5R)-carbapen-2-em-3-carboxylate
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
-
-
?
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Pectobacterium carotovorum
Q9XB61
-
-
Purification (Commentary)
Commentary
Organism
-
Pectobacterium carotovorum
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ATP + (2R,5R)-5-carboxymethylproline
low activity, kcat/Km is 2% compared to the value for (2S,5S)-5-carboxymethylproline
721590
Pectobacterium carotovorum
?
-
-
-
?
ATP + (2S,5R)-5-carboxymethylproline
low activity, kcat/Km is 2% compared to the value for (2S,5S)-5-carboxymethylproline
721590
Pectobacterium carotovorum
?
-
-
-
?
ATP + (2S,5S)-5-carboxymethylproline
the enzyme is involved in the biosynthesis of the carbapenem beta-lactam antibiotic (5R)-carbapen-2-em-3-carboxylate
721590
Pectobacterium carotovorum
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
-
-
-
?
ATP + (2S,5S)-5-carboxymethylproline
the kinetic mechanism is bi-ter where ATP is the first substrate to bind followed by (2S,5S)-5-carboxymethyl proline and diphosphate is the last product released. Low activity with (2S,5R)-5-carboxymethylproline or (2R,5R)-5-carboxymethylproline
721590
Pectobacterium carotovorum
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
-
-
-
?
Subunits
Subunits
Commentary
Organism
homodimer
2 * 56000, calculated from sequence
Pectobacterium carotovorum
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
22
-
assay at
Pectobacterium carotovorum
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.122
-
(2R,5R)-5-carboxymethylproline
pH 7.8, 22°C
Pectobacterium carotovorum
0.2
-
(2S,5R)-5-carboxymethylproline
pH 7.8, 22°C
Pectobacterium carotovorum
0.28
-
(2S,5S)-5-carboxymethylproline
pH 7.8, 22°C
Pectobacterium carotovorum
0.28
-
ATP
pH 7.8, 22°C
Pectobacterium carotovorum
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.8
-
assay at
Pectobacterium carotovorum
Ki Value [mM]
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
0.005
-
diphosphate
pH 7.8, 22°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(slope)
Pectobacterium carotovorum
0.012
-
diphosphate
pH 7.8, 22°C, variable substrate: ATP, Ki(slope)
Pectobacterium carotovorum
0.019
-
diphosphate
pH 7.8, 22°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(intercept)
Pectobacterium carotovorum
0.17
-
AMP
pH 7.8, 22°C, variable substrate: ATP, Ki(intercept)
Pectobacterium carotovorum
0.28
-
AMP
pH 7.8, 22°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(intercept)
Pectobacterium carotovorum
1.1
-
(3S,5S)-carbapenam 3-carboxylate
pH 7.8, 22°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(intercept)
Pectobacterium carotovorum
1.3
-
(3S,5S)-carbapenam 3-carboxylate
pH 7.8, 22°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(slope); pH 7.8, 22°C, variable substrate: ATP, Ki(slope)
Pectobacterium carotovorum
90
-
L-proline
pH 7.8, 22°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(slope)
Pectobacterium carotovorum
207
-
L-proline
pH 7.8, 22°C, variable substrate: ATP, Ki(intercept)
Pectobacterium carotovorum
Cloned(Commentary) (protein specific)
Commentary
Organism
overexpression of the plasmid pET24a/carA transformed in Escherichia coli BL21(DE3) cells
Pectobacterium carotovorum
Inhibitors (protein specific)
Inhibitors
Commentary
Organism
Structure
(3S,5S)-carbapenam 3-carboxylate
-
Pectobacterium carotovorum
AMP
uncompetitive inhibition versus both substrates
Pectobacterium carotovorum
diphosphate
the competitive inhibition exhibited by diphosphate versus ATP allows the assignment of diphosphate as the last product released
Pectobacterium carotovorum
L-proline
the uncompetitive nature of the inhibition of the dead-end inhibitor, L-proline, versus ATP is consistent with a mechanism with ordered substrate binding where ATP binds first followed by (2S,5S)-carboxymethylproline
Pectobacterium carotovorum
Ki Value [mM] (protein specific)
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
0.005
-
diphosphate
pH 7.8, 22°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(slope)
Pectobacterium carotovorum
0.012
-
diphosphate
pH 7.8, 22°C, variable substrate: ATP, Ki(slope)
Pectobacterium carotovorum
0.019
-
diphosphate
pH 7.8, 22°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(intercept)
Pectobacterium carotovorum
0.17
-
AMP
pH 7.8, 22°C, variable substrate: ATP, Ki(intercept)
Pectobacterium carotovorum
0.28
-
AMP
pH 7.8, 22°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(intercept)
Pectobacterium carotovorum
1.1
-
(3S,5S)-carbapenam 3-carboxylate
pH 7.8, 22°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(intercept)
Pectobacterium carotovorum
1.3
-
(3S,5S)-carbapenam 3-carboxylate
pH 7.8, 22°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(slope); pH 7.8, 22°C, variable substrate: ATP, Ki(slope)
Pectobacterium carotovorum
90
-
L-proline
pH 7.8, 22°C, variable substrate: (2S,5R)-carboxymethylproline, Ki(slope)
Pectobacterium carotovorum
207
-
L-proline
pH 7.8, 22°C, variable substrate: ATP, Ki(intercept)
Pectobacterium carotovorum
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.11
-
ATP
pH 7.8, 22°C
Pectobacterium carotovorum
0.23
-
(2S,5S)-5-carboxymethylproline
pH 7.8, 22°C
Pectobacterium carotovorum
4.6
-
(2R,5R)-5-carboxymethylproline
pH 7.8, 22°C
Pectobacterium carotovorum
7
-
(2S,5R)-5-carboxymethylproline
pH 7.8, 22°C
Pectobacterium carotovorum
Molecular Weight [Da] (protein specific)
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
56000
-
2 * 56000, calculated from sequence
Pectobacterium carotovorum
119000
-
native PAGE
Pectobacterium carotovorum
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ATP + (2S,5S)-5-carboxymethylproline
Pectobacterium carotovorum
the enzyme is involved in the biosynthesis of the carbapenem beta-lactam antibiotic (5R)-carbapen-2-em-3-carboxylate
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
-
-
?
Purification (Commentary) (protein specific)
Commentary
Organism
-
Pectobacterium carotovorum
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ATP + (2R,5R)-5-carboxymethylproline
low activity, kcat/Km is 2% compared to the value for (2S,5S)-5-carboxymethylproline
721590
Pectobacterium carotovorum
?
-
-
-
?
ATP + (2S,5R)-5-carboxymethylproline
low activity, kcat/Km is 2% compared to the value for (2S,5S)-5-carboxymethylproline
721590
Pectobacterium carotovorum
?
-
-
-
?
ATP + (2S,5S)-5-carboxymethylproline
the enzyme is involved in the biosynthesis of the carbapenem beta-lactam antibiotic (5R)-carbapen-2-em-3-carboxylate
721590
Pectobacterium carotovorum
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
-
-
-
?
ATP + (2S,5S)-5-carboxymethylproline
the kinetic mechanism is bi-ter where ATP is the first substrate to bind followed by (2S,5S)-5-carboxymethyl proline and diphosphate is the last product released. Low activity with (2S,5R)-5-carboxymethylproline or (2R,5R)-5-carboxymethylproline
721590
Pectobacterium carotovorum
AMP + diphosphate + (3S,5S)-carbapenam 3-carboxylate
-
-
-
?
Subunits (protein specific)
Subunits
Commentary
Organism
homodimer
2 * 56000, calculated from sequence
Pectobacterium carotovorum
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
22
-
assay at
Pectobacterium carotovorum
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.122
-
(2R,5R)-5-carboxymethylproline
pH 7.8, 22°C
Pectobacterium carotovorum
0.2
-
(2S,5R)-5-carboxymethylproline
pH 7.8, 22°C
Pectobacterium carotovorum
0.28
-
(2S,5S)-5-carboxymethylproline
pH 7.8, 22°C
Pectobacterium carotovorum
0.28
-
ATP
pH 7.8, 22°C
Pectobacterium carotovorum
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.8
-
assay at
Pectobacterium carotovorum
General Information
General Information
Commentary
Organism
physiological function
the enzyme is involved in the biosynthesis of the carbapenem beta-lactam antibiotic (5R)-carbapen-2-em-3-carboxylate
Pectobacterium carotovorum
General Information (protein specific)
General Information
Commentary
Organism
physiological function
the enzyme is involved in the biosynthesis of the carbapenem beta-lactam antibiotic (5R)-carbapen-2-em-3-carboxylate
Pectobacterium carotovorum
KCat/KM [mM/s]
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
0.026
-
(2R,5R)-5-carboxymethylproline
pH 7.8, 22°C
Pectobacterium carotovorum
0.027
-
(2S,5R)-5-carboxymethylproline
pH 7.8, 22°C
Pectobacterium carotovorum
1.2
-
(2S,5S)-5-carboxymethylproline
pH 7.8, 22°C
Pectobacterium carotovorum
2.5
-
ATP
pH 7.8, 22°C
Pectobacterium carotovorum
KCat/KM [mM/s] (protein specific)
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
0.026
-
(2R,5R)-5-carboxymethylproline
pH 7.8, 22°C
Pectobacterium carotovorum
0.027
-
(2S,5R)-5-carboxymethylproline
pH 7.8, 22°C
Pectobacterium carotovorum
1.2
-
(2S,5S)-5-carboxymethylproline
pH 7.8, 22°C
Pectobacterium carotovorum
2.5
-
ATP
pH 7.8, 22°C
Pectobacterium carotovorum
Other publictions for EC 6.3.3.6
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)
743905
Hamed
-
Stereoselective production of ...
Pectobacterium carotovorum
ACS Catal.
7
1279-1285
2017
-
1
1
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
5
-
-
-
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
727703
Tahlan
Origins of the beta-lactam rin ...
Pectobacterium carotovorum, Streptomyces cattleya
J. Antibiot.
66
401-410
2013
-
-
-
-
-
-
-
-
-
-
-
2
-
3
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
721613
Raber
A conserved tyrosyl-glutamyl c ...
Pectobacterium carotovorum
Biochemistry
48
4959-40971
2009
-
-
-
-
8
-
-
8
-
-
-
-
-
1
-
-
-
-
-
-
-
-
1
-
1
-
-
8
-
-
-
-
-
-
-
-
-
-
-
-
8
-
-
-
-
8
-
-
-
-
-
-
-
-
-
-
-
-
1
-
1
-
-
8
-
-
-
-
-
-
-
-
8
8
721605
Arnett
Rate-limiting steps and role o ...
Pectobacterium carotovorum
Biochemistry
46
9337-9345
2007
-
-
1
-
2
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
1
-
1
-
-
1
1
-
-
-
-
-
-
-
-
1
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
1
-
1
-
-
1
1
-
-
-
-
-
-
-
1
1
721590
Gerratana
Inhibition and alternate subst ...
Pectobacterium carotovorum
Biochemistry
42
7836-7847
2003
-
-
1
-
-
-
4
4
-
-
2
1
-
4
-
-
1
-
-
-
-
-
4
1
1
-
-
4
1
-
-
-
9
-
-
-
-
1
-
-
-
-
-
4
9
4
-
-
2
1
-
-
-
1
-
-
-
-
4
1
1
-
-
4
1
-
-
-
-
1
1
-
4
4
722636
Miller
Crystal structure of carbapena ...
Pectobacterium carotovorum
J. Biol. Chem.
278
40996-41002
2003
-
-
1
1
-
-
-
-
-
1
2
1
-
1
-
-
1
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
1
-
-
-
-
-
-
-
1
2
1
-
-
-
1
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-