Information on EC 1.13.11.15 - 3,4-dihydroxyphenylacetate 2,3-dioxygenase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.13.11.15
-
RECOMMENDED NAME
GeneOntology No.
3,4-dihydroxyphenylacetate 2,3-dioxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
3,4-dihydroxyphenylacetate + O2 = 2-hydroxy-5-carboxymethylmuconate semialdehyde
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
4-hydroxyphenylacetate degradation
Tyrosine metabolism
-
-
Microbial metabolism in diverse environments
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-
SYSTEMATIC NAME
IUBMB Comments
3,4-dihydroxyphenylacetate:oxygen 2,3-oxidoreductase (decyclizing)
An iron protein.
CAS REGISTRY NUMBER
COMMENTARY hide
37256-56-7
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
Arthrobacter synephrinum
-
-
-
Manually annotated by BRENDA team
weak activity
-
-
Manually annotated by BRENDA team
C
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
weak activity
-
-
Manually annotated by BRENDA team
weak activity
-
-
Manually annotated by BRENDA team
gene hpaD
-
-
Manually annotated by BRENDA team
CBS3
-
-
Manually annotated by BRENDA team
weak activity
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
-
In mutant Y257F, steps of the catalytic cycle are slowed by as much as 100fold by the mutation compared to the wild-type enzyme due to failure of mutant Y257F to facilitate the observed distortion of the bound 3,4-dihydroxyphenylacetate that is proposed to promote transfer of one electron to O2, Steady-state and transient kinetic analyses
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2,3-dihydroxybenzoate + O2
CO2 + alpha-hydroxymuconic semialdehyde
show the reaction diagram
-
-
mutant H200F produces alpha-carboxy-cis,cis-muconic acid
-
?
3,4 dihydroxyphenylacetate + O2
1-carboxymethyl-4-hydroxy-cis-muconic semialdehyde
show the reaction diagram
-
-
-
-
?
3,4-dihydroxybenzoic acid + O2
2-hydroxy-5-carboxymuconate semialdehyde
show the reaction diagram
3,4-dihydroxymandelate + O2
?
show the reaction diagram
3,4-dihydroxyphenylacetate + O2
1-carboxymethyl-4-hydroxy-cis-muconic semialdehyde
show the reaction diagram
-
-
-
-
?
3,4-dihydroxyphenylacetate + O2
2-hydroxy-5-5carboymethylmuconate semialdehyde
show the reaction diagram
3,4-dihydroxyphenylacetate + O2
2-hydroxy-5-carboxymethyl-cis,cis-muconate semialdehyde
show the reaction diagram
-
-
-
-
?
3,4-dihydroxyphenylacetate + O2
2-hydroxy-5-carboxymethylmuconate semialdehyde
show the reaction diagram
3,4-dihydroxyphenylacetate + O2
2-hydroxy-5-carboymethylmuconate semialdehyde
show the reaction diagram
3,4-dihydroxyphenylacetate + O2
?
show the reaction diagram
3,4-dihydroxyphenylpropionate + O2
2-hydroxy-5-carboxyethylmuconate semialdehyde
show the reaction diagram
3-(3,4-dihydroxyphenyl)-DL-alanine + O2
(2E,4Z)-7-amino-5-formyl-2-hydroxyocta-2,4-dienedioic acid
show the reaction diagram
-
-
-
-
?
4-nitrocatechol + O2
?
show the reaction diagram
N-formyl-3-(3,4-dihydroxyphenyl)-DL-alanine + O2
(2E,4Z)-5-formyl-7-(formylamino)-2-hydroxyocta-2,4-dienedioic acid
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
3,4 dihydroxyphenylacetate + O2
1-carboxymethyl-4-hydroxy-cis-muconic semialdehyde
show the reaction diagram
-
-
-
-
?
3,4-dihydroxyphenylacetate + O2
2-hydroxy-5-5carboymethylmuconate semialdehyde
show the reaction diagram
3,4-dihydroxyphenylacetate + O2
2-hydroxy-5-carboxymethylmuconate semialdehyde
show the reaction diagram
additional information
?
-
-
LB400 cells grown with 3-hydroxyphenylacetate degrade homoprotocatechuate and show homoprotocatechuate 2,3-dioxygenase activity
-
-
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ascorbate
-
activates at 0.5 mM
Fe
-
contains an active site Fe(II)
Fe(NH4)2(SO4)2
-
activates at 0.5 mM, activation is enhanced by 10 mM DTT
Fe3+
-
nonphysiological metal, incorporated in Fe-MndD
Manganese
Mg2+
-
1.1-1.3 gatom of Mg2+ per mol of enzyme, essential for activity
Mn
-
wild-type enzyme amd mutant enzymes H200A and H200Q: 0.6 Mn per monomer. Mutant enzyme H200E: 0.3 Mn per monomer. Mutent enzyme H200N: 0.4 MN per monomer
NO
-
NO binding to a non-heme enzyme containing manganese allows examination of the factors governing the formation and detection of the MIII-O2.- species in all forms of th enzyme. NO, and presumably O2, binding is sensitive to both the nature of the catecholic substrate present and the nature of the active-site amino acid residue at position 200, spectral analysis, overview
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
3,4-dichlorophenylacetate
-
5 mM, 50% inhibition
3,4-dihydroxybenzoate
3,4-dihydroxycinnamate
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-
3,4-Dihydroxyphenylpropionate
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4-methylcatechol
-
-
4-nitrocatechol
5,5'-dithiobis-(2-nitrobenzoate)
Arthrobacter synephrinum
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1 mM, complete inhibition
8-hydroxyquinoline
air
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initially high activity of Fe-MndD, becomes inactivtated within one h
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Copper salicylate
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0.166 mM, rate of oxygen uptake is decreased by 38%, rate of product formation is diminished by 58%
Cu2+
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0.01 mM, 80% inhibition
dithiothreitol
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0.1 mM, 13% inhibition
DTT
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destabilizes the enzyme
EDTA
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destabilizes the enzyme
ferricyanide
Ferrous ammonium sulfate
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0.1 mM, 32% inhibition
K3Fe(CN)6
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10 mM, 35% inhibition
m-hydroxyphenylacetate
-
-
Mn2+
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slight inhibition
Na2SO4
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destabilizes the enzyme
NaN3
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slightly destabilizes the enzyme
p-hydroxymercuribenzoate
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0.1 mM, 13% inhibition
p-hydroxyphenylacetate
-
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succinate semialdehyde
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5 mM 65% inhibition
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Sodium ascorbate
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1 mM, revers purification inactivation of Fe-MndD, about 30-35% higher catalytic activity than the purified enzyme
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.074 - 0.45
3,4-dihydroxybenzoic acid
0.5
3,4-dihydroxymandelate
Arthrobacter synephrinum
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-
0.0004 - 0.067
3,4-Dihydroxyphenylacetate
0.0037
3,4-Dihydroxyphenylpropionic acid
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32C
0.015
4-nitrocatechol
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pH 7.5, 22C
0.035
DL-3,4-dihydroxymandelic acid
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32C
0.037 - 0.062
O2
additional information
additional information
-
Michaelis-Menten steady-state kinetics under air-saturated conditions. The rate determining steps of the PaDHPAO reaction at temperatures above and below 35C may be different
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.01 - 0.02
2,3-Dihydroxybenzoate
0.067 - 63.5
3,4-Dihydroxyphenylacetate
0.05 - 0.4
4-nitrocatechol
additional information
additional information
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
900 - 1100
3,4-Dihydroxyphenylacetate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.3
3,4-dihydroxybenzoate
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0.3
3,4-dihydroxycinnamate
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1
3,4-Dihydroxyphenylpropionate
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1.5
4-methylcatechol
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0.5
4-nitrocatechol
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3.8
catechol
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0.27
m-hydroxyphenylacetate
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1.8
p-hydroxyphenylacetate
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-
additional information
additional information
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Ki-values for 4-nitrocatechol for the H200A, H200E, H200N and H200Q variants are between 0.015 and 0.025 mM
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2.51
Arthrobacter synephrinum
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-
6.2
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recombinant enzyme
44.3
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purified recombinant enzyme, pH 7.5, 25C
additional information
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-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 7.5
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assay at
6.9
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substrate 4-nitrocatechol
7 - 8
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mutant enzyme H200N and H200Q
7.2
Arthrobacter synephrinum
-
-
7.2 - 7.8
-
-
8 - 9
-
wild-type enzyme
8.4
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substrate 3,4-dihydroxyphenylacetate
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 9.5
-
activity range, profile overview. pH-rate profile of enzyme PaDHPAO reaction shows a bell-shaped plot that exhibits a maximum activity at pH 7.5 with two pKa values of pH 6.5 and pH 8.9
7.1 - 9.3
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78% of maximal activity at pH 7.1 and 9.3
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 22
-
assay at 4C or 22C
22
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assay at room temperature
50
Arthrobacter synephrinum
-
-
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 70
-
activity range, profile overview. The enzyme activity increases as temperature increases up to 55C and then decreases at higher temperatures. The rate determining steps of the PaDHPAO reaction at temperatures above and below 35C may be different
40 - 64
-
40C: about 40% of maximal activity, 64C: about 75% of maximal activity
PDB
SCOP
CATH
ORGANISM
UNIPROT
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25500
-
4 * 25500, SDS-PAGE
31000
-
x * 31000, SDS-PAGE
33000
-
gel filtration
35000
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4 * 35000, SDS-PAGE
36000
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4 * 36000, SDS-PAGE
42500
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4 * 42500, or 6 * 42500, SDS-PAGE; 6 * 42500, or 42500, SDS-PAGE
102000
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gel filtration
106000
-
gel filtration
112200
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recombinant enzyme, gel filtration
120000
-
equilibrium sedimentation
138000
-
gel filtration
140000
-
gel filtration
220000
-
equilibrium sedimentation
282000
Arthrobacter synephrinum
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexamer
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6 * 42500, or 42500, SDS-PAGE
homotetramer
tetramer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystallizations of wild-type FeHPCD and mutant Y257F with bound substrates 3,4-dihydroxyphenylacetate and 4-nitrocatechol, hanging drop vapour diffusion method, in either 13% PEG 6000, 0.1 M calcium chloride, 0.1 M Tris-HCl, pH 6.5-7.4 or in 18% PEG 8000, 0.1 M calcium acetate, 0.1 M sodium cacodylate, pH 6.5, at 20C, cryoprotectant solution containing either 20-25% PEG400 or 20% glycerol in the mother liquor solution, X-ray diffraction structure determination and analysis at 1.50-1.60 A resolution
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
-
most stable at
439614
7.5
-
maximal stability at
439626
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0
-
pH 7.5, 0.1 M sodium potassium phosphate, 1 mM DTT, complete loss of activity after 48 h
40
-
half-life: 13 min
50
Arthrobacter synephrinum
-
30 min, stable
55
-
5 min, stable
63
-
2 h, 5% loss of activity
88
-
10 min, 50% loss of activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
dialysis against buffer for several hours at 0C, no appreciable loss of activity
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Fe-HPCD stable in air for at least several days
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inactivation by 6 M guanidine hydrochloride
Arthrobacter synephrinum
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stabilization in presence of low concentrations of acetone or ethanol
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
10C, lyophilized preparations of the enzyme, 8 months, stable
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room temperature, little loss of activity after 3 months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
partial
recombinant enzyme from Escherichia coli
-
recombinant soluble enzyme 2.5fold from Escherichia coli strain BL21 (DE3) by anion exchange chromatography, ultrafiltration, hydrophobic interaction chromatography, ultrafiltration, and gel filtration
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recombinant wild-type and mutant Y257F enzymes by a procedure including anion exchange chromatography
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wild-type and mutant enzymes
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wild-type enzyme and H200 mutants
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli BL21
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expression in Escherichia coli DH5alpha
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gene dhpao, DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant expression in Escherichia coli strain BL21 (DE3)
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gene hpaD, gene cluster hpaG1G2EDFHI encodes the homoprotocatechuate pathway is located at C2 in strain LB400 genome, quantitative expression analysis
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overexpression in Escherichia coli
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recombinant expression of wild-type and mutant Y257F enzymes
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wild-type and mutant enzymes expressed in Escherichia coli
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
induction of fumarylacetoacetate hydrolase HmgB in Burkholderia xenovorans LB400 during growth on hydroxyphenylacetates
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LB400 cells grown with 3-hydroxyphenylacetate and 4-hydroxyphenylacetate both show induction of the homoprotocatechuate pathway, they degrade homoprotocatechuate and show homoprotocatechuate 2,3-dioxygenase activity. The hmgA1 gene is upregulated by 3-hydroxyphenylacetate in strain LB400. The expression of the hpaD gene, encoding HPC 2,3-dioxygenase, is induced in 3-hydroxyphenylacetate and 4-hydroxyphenylacetate grown-cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E266Q
-
specific activity is less than 0.1% of that of the wild-type enzyme, binds 33% of the wild-type level of manganese
H155A
-
specific activity is less than 0.1% of that of the wild-type enzyme, binds 0.4% of the wild-type level of manganese
H200A
-
KM-value for 3,4-dihydroxyphenylacetate is 1.75fold lower than wild-type value, kcat for 3,4-dihydroxyphenylacetate is 69fold lower than wild-type value
H200E
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KM-value for 3,4-dihydroxyphenylacetate is 1.2fold lower than wild-type value, kcat for 3,4-dihydroxyphenylacetate is more than 276fold lower than wild-type value, Mn content per monomer is 2fold lower than in wild-type enzyme
H200N
-
KM-value for 3,4-dihydroxyphenylacetate is identical to wild-type value, kcat for 3,4-dihydroxyphenylacetate is 1.73fold lower than wild-type value, Mn content per monomer is 65% of wild-type value
H200Q
-
KM-value for 3,4-dihydroxyphenylacetate is 1.2fold lower than wild-type value, kcat for 3,4-dihydroxyphenylacetate is 1.3fold lower than wild-type value
H214A
-
specific activity is less than 0.1% of that of the wild-type enzyme, binds 1.8% of the wild-type level of manganese
H42A
-
97% of wild-type activity at 18C, 30% of wild-type activity at 37C
E266Q
-
specific activity is less than 0.1% of that of the wild-type enzyme, binds 33% of the wild-type level of manganese
-
H155A
-
specific activity is less than 0.1% of that of the wild-type enzyme, binds 0.4% of the wild-type level of manganese
-
H200A
-
KM-value for 3,4-dihydroxyphenylacetate is 1.75fold lower than wild-type value, kcat for 3,4-dihydroxyphenylacetate is 69fold lower than wild-type value
-
H200E
-
KM-value for 3,4-dihydroxyphenylacetate is 1.2fold lower than wild-type value, kcat for 3,4-dihydroxyphenylacetate is more than 276fold lower than wild-type value, Mn content per monomer is 2fold lower than in wild-type enzyme
-
H200N
-
KM-value for 3,4-dihydroxyphenylacetate is identical to wild-type value, kcat for 3,4-dihydroxyphenylacetate is 1.73fold lower than wild-type value, Mn content per monomer is 65% of wild-type value
-
H200Q
-
KM-value for 3,4-dihydroxyphenylacetate is 1.2fold lower than wild-type value, kcat for 3,4-dihydroxyphenylacetate is 1.3fold lower than wild-type value
-
H214A
-
specific activity is less than 0.1% of that of the wild-type enzyme, binds 1.8% of the wild-type level of manganese
-
H42A
-
97% of wild-type activity at 18C, 30% of wild-type activity at 37C
-
H200A
-
Km-value for 3,4-dihydroxyphenylacetate is 6fold lower than wild-type value, kcat for 3,4-dihydroxyphenylacetate is 33.3fold lower than wild-type value
H200E
-
Km-value for 3,4-dihydroxyphenylacetate is 75fold lower than wild-type value, kcat for 3,4-dihydroxyphenylacetate is 11.1fold lower than wild-type value
H200Q
-
Km-value for 3,4-dihydroxyphenylacetate is similar to wild-type value, kcat for 3,4-dihydroxyphenylacetate is 2.5fold lower than wild-type value
additional information
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