Information on EC 3.4.21.46 - complement factor D

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.4.21.46
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RECOMMENDED NAME
GeneOntology No.
complement factor D
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
selective cleavage of Arg-/-Lys bond in complement factor B when in complex with complement subcomponent C3b or with cobra venom factor
show the reaction diagram
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-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
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endopeptidase
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CAS REGISTRY NUMBER
COMMENTARY hide
37213-56-2
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
rainbow trout
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
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in humans neither MASP-1 nor MASP-3 are required for alternative pathway function. During MASP-1/-3 deficiency significant alternative pathway activity remains, indicating that mature factor D is present in the absence of MASP-1/-3
physiological function
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
Ac-Lys-Arg-4-nitroanilide + H2O
Ac-Lys-Arg + 4-nitroaniline
show the reaction diagram
-
-
-
-
?
acetyl-Gly-L-Lys methyl ester + H2O
?
show the reaction diagram
-
-
-
-
?
Benzoyl-L-Arg methyl ester + H2O
Benzoyl-L-Arg + methanol
show the reaction diagram
-
-
-
-
?
benzyloxycarbonyl-Arg-benzyl thioester + H2O
?
show the reaction diagram
-
-
-
-
?
benzyloxycarbonyl-Lys-Arg-isobutyl thioester + H2O
?
show the reaction diagram
-
-
-
-
?
benzyloxycarbonyl-Lys-benzyl thioester + H2O
?
show the reaction diagram
-
-
-
-
?
complement component C3bB + H2O
?
show the reaction diagram
complement factor B + H2O
Bb fragment + Ba fragment
show the reaction diagram
factor B + H2O
Ba + Bb
show the reaction diagram
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-
smaller and larger cleavage subunit of factor B, respectively
-
?
factor B + H2O
Bb fragment + Ba fragment
show the reaction diagram
N-Acetyl-L-Tyr ethyl ester + H2O
?
show the reaction diagram
-
-
-
-
?
N-benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide + H2O
?
show the reaction diagram
-
-
-
-
?
Nalpha-acetyl-L-Arg methyl ester + H2O
?
show the reaction diagram
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-
-
-
?
Nalpha-acetyl-L-Lys methyl ester + H2O
?
show the reaction diagram
-
-
-
-
?
Nalpha-Benzoyl-L-Arg ethyl ester + H2O
?
show the reaction diagram
-
-
-
-
?
Nalpha-tosyl-L-Lys methyl ester + H2O
?
show the reaction diagram
-
-
-
-
?
Nepsilon-carbobenzoxy-L-Lys methyl ester + H2O
?
show the reaction diagram
-
-
-
-
?
p-tosyl-L-Arg methyl ester + H2O
?
show the reaction diagram
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-
-
-
?
pro-convertase C3bB + H2O
active C3 convertase C3bBb + ?
show the reaction diagram
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-
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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
complement component C3bB + H2O
?
show the reaction diagram
complement factor B + H2O
Bb fragment + Ba fragment
show the reaction diagram
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?
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
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required
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3,4-dichloroisocoumarin
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mechanism-based inactivation
anti-factor D Fab fragment
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diisopropyl fluorophosphate
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isatoic anhydride
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-
additional information
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human factor D is a self-inhibited thrombin-like serine proteinase. Ser199 of the self-inhibitory loop blocks the formation of the canonical active configurations
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
complement factor B
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the enzyme factor D is activated by its substrate factor B through interactions outside the active site. The substrate-binding, or exosite, region displays a well defined and rigid conformation
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additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.95 - 11.44
Benzyloxycarbonyl-Lys-thiobenzyl ester
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.3 - 21.3
Benzyloxycarbonyl-Lys-thiobenzyl ester
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.1 - 7.2
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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low levels of vegf-D mRNA
Manually annotated by BRENDA team
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PDGF-D is widely expressed in most neointimas in arteries exhibiting the chronic arteriopathy of chronic allograft nephropathy and is only weakly expressed in a small proportion of sclerotic arteries. The neointimal cells expressing PDGF-D are alpha-smooth muscle actin-expressing cells, but not infiltrating macrophages or endothelial cells
Manually annotated by BRENDA team
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PDGF-D high-expressing breast cancer cell line SUM-149 is more invasive compared to low-expressing cell lines
Manually annotated by BRENDA team
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unfertilized
Manually annotated by BRENDA team
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hatchling; transcript levels steadily increased from day 28 post-fertilization to hatch
Manually annotated by BRENDA team
high mRNA expression determined by real-time RT-PCR
Manually annotated by BRENDA team
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low levels of vegf-D mRNA
Manually annotated by BRENDA team
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low PDGF-D expressing breast cancer cell line is less invasive
Manually annotated by BRENDA team
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PDGF-D expressing breast cancer cell lines are more invasive compared to non-expressing cell lines
Manually annotated by BRENDA team
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low PDGF-D expressing breast cancer cell line is less invasive
Manually annotated by BRENDA team
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low levels of vegf-D mRNA
Manually annotated by BRENDA team
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5- to 8fold higher amounts of vegf-D mRNA as the other gastric cell lines
Manually annotated by BRENDA team
moderate mRNA expression determined by real-time RT-PCR
Manually annotated by BRENDA team
moderate mRNA expression determined by real-time RT-PCR
Manually annotated by BRENDA team
moderate mRNA expression determined by real-time RT-PCR
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
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occasionally in peritumoral stroma
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Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
21000
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Westernblot
22900
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equilibrium sedimentation
23000
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x * 23000, SDS-PAGE
24000
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x * 24000, SDS-PAGE
24376
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x * 24376, calculation from nucleotide sequence
25000
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x * 25000, SDS-PAGE
30020
deduced from cDNA
290000
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SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
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SDS-PAGE and immunoblotting. VEGF-DDeltaNDeltaC homodimer is antiparallel and has an elongated shape but is flat at the central part. It has three subunit interface cysteines C25, C44 and C53
monomer
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
no modification
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no potential N-glycosylation site, no carbohydrate is present in the purified protein
proteolytic modification
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure of profactor D
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purified enzyme in complex with anti-factor D Fab fragment, sitting drop vapor diffusion method, 30 mg/ml protein in 20 mM HEPES, pH 7.2, and 200 mM NaCl, is mixed with an equal volume of crystallization buffer containing 0.1 M Tris-HCl, pH 8.5, 0.2 M ammonium phosphate, 50% 2-methyl-2,4-pentanediol, and 0.01 M hexamine cobalt(III) chloride, 6 days, 4°C, X-ray diffraction structure determination and analysis at 2.4 A resolution
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S183A mutant protein in complex with pro-convertase C3bB
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structure of diisopropyl fluorophosphate-inhibited factor D
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structure of native factor D and a complex formed with isatoic anhydride inhibitor
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wild type and mutant factor D, expression in CHO cells
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wild-type enzyme, mutants R202A and S81Y/T198S/S199W, and enzyme mutant S183A in ternary complex with C3bB, hanging drop vapour diffusion technique, mixing of 10 mg/ml protein solution with reservoir solution consisting of 15% w/v PEG 6000, 50 mM MES-NaOH pH 6.0, 18°C, 6 days, X-ray diffraction structure determination and analysis at 1.8 A, 2.8 A, 2.0 A, and 3.5 A resolution, respectively, molecular replacement and ensemble refinement
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purified enzyme in complex with anti-factor D Fab fragment, sitting drop vapor diffusion method, 20 mg/ml protein in 20 mM HEPES, pH 7.2, and 200 mM NaCl, is mixed with an equal volume of crystallization buffer containing 0.1 M MES, pH 6.5, 25% PEG monomethyl ether 550, 0.01 M zinc sulfate, and 3% 6-aminohexanoic acid, 1 day, 19°C, X-ray diffraction structure determination and analysis at 2.3 A resolution
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
freezing and thawing causes approximately 30% loss of activity
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C, no aggregation occurs, stable
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4°C, 0.1 M Tris-HCl, 0.2 M NaCl, 2 mM EDTA, pH 8.0, 0.02% w/v NaN3, several months, about 10% loss of activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cation-exchange chromatography,gelfiltration
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enzyme and inhibitor by gel filtration
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gel filtration
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recombinant soluble wild-type and mutant enzymes from HEK-293-E cells by cation exchange chromatography and gel filtration
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Sephacryl S-100 and DEAE Sepharose fast flow column chromatography, 4°C
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VEGF-DDELTANDELTAC and the three cysteine mutants purified by metal affinity chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
baculoviral VEGF-D or adenoviral VEGF-D expressed in the rabbit eye. VEGF-D expression shows a similar pattern in the retina and retinal pigment epithelium layer after baculoviral and adenoviral transduction. At 6 days after gene transfer, both viruses show dose-dependent increase in the expression of human VEGF-D in the vitreous humour
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Df-deficient mice show protection against the development of proliferative renal disease
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expressed in breast cancer cell lines MDA-MB-468 and MCF-7 cells
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expressed in HEK293-E cells
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factor D gene expression is upregulated by western diet
gastric cells transiently transfected with the pGL3-based vegf-D reporter gene construct vegf-D(-2011/+110), which comprises nucleotides -2011/+110 of the human vegf-D gene
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mutants transiently transfected into 293T cells. The native VEGF-DDELTANDELTAC and mutants expressed in a larger scale in insect cells using BVboostFG baculovirus expression system
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transient expression of wild-type and mutant enzymes in HEK-293-E cells from pUPE.05.05 expression vectors, the recombinant proteins are secreted
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
deguelin, a rotenoid of the flavonoid family, significantly down-regulates the expression of VEGF-D both at the mRNA and protein level in VEGF-D transfected LL/2 Lewis lung cells
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expression of PoDAK increases in kidney, liver, spleen upon infection with viral hemorrhagic septicemia virus or bacterial infection with Streptococcus iniae
in lung cancer cell lines, interleukin-7/interleukin-7 receptor increase the expression of VEGF-D and phosphorylation of c-Fos/c-Jun, induce c-Fos and c-Jun heterodimer formation, and enhance c-Fos/c-Jun DNA binding activity to regulate VEGF-D expression
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overexpression of PDGF-D in PDGF-D low expressing breast cancer cell lines MDA-MB-468 and MCF-7 cells by cDNA transfection shows increased cell proliferation. PDGF-D overexpression is positively correlated with the expression of Notch-1 and Jagged-1, and the expression of mesenchymal markers (Vimentin and ZEB-2) with concomitant decreased expression of epithelial marker E-cadherin. PDGF-D over-expression leads to increased DNA binding activity of NF-kappaB
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silencing the expression of PDGF-D by siRNA in PDGF-D high expressing MDA-MB-231 and SUM-149 cells shows decreased cell proliferation and increased apoptosis
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C25A
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has increased activity compared to the native VEGF-DDELTANDELTAC. Efficiently binds to the soluble receptor VEGFR-2/IgGFc. The C25A mutant behaves mainly as a monomeric protein on SDS-PAGE under reducing conditions but as a dimeric protein under non-reducing conditions. It induces VEGFR-2 Tyr-1175 phosphorylation
C25A/P43S
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has biological activity comparable to that of the native protein in Ba/F3-VEGFR-2 cells
C25I
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is mainly in a presumably covalently bound dimeric form
C25L
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has increased activity compared to the native VEGF-DDELTANDELTAC, the C25L mutant is the most active mutant and is mainly in a presumably covalently bound dimeric form, has highest affinity to bind soluble receptor VEGFR-2/IgGFc. It induces VEGFR-2 Tyr-1175 phosphorylation
C25V
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is mainly in a presumably covalently bound dimeric form
C44A
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does not bind to the soluble receptor VEGFR-2/IgGFc
C53A
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does not bind the soluble receptors VEGFR-2/IgGFc and VEGFR-3/IgGFc; does not bind to the soluble receptor VEGFR-2/IgGFc
G51C
-
can not induce cell survival, has increased dimer to monomer ratio
H133A
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catalytically inactive
P43S
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can not induce cell survival
R115C
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activity like wild type
R157A
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catalytically inactive
R22I
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can not induce cell survival
R22I/C25L
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has biological activity comparable to that of the native protein in Ba/F3-VEGFR-2 cells, has increased dimer to monomer ratio
R22L
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can not induce cell survival
R22L/C25L
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has biological activity comparable to that of the native protein in Ba/F3-VEGFR-2 cells, has increased dimer to monomer ratio
S81Y/T198S/S199W
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site-directed mutagenesis, the mutation causes steric hindrance of Trp199 resulting in pronounced rearrangement of the proteinase active-site region, structure analysis
S94Y
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6fold increase in turnover number
S94Y/T214/S215W
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the triple mutant has a turnover number that is 16fold higher than that of wild type factor D
T214S/S215W
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more than 16fold incerease of the ratio of turnover number/KM-value
T638G
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factor D deficient mutant
T640C
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factor D deficient mutant
V203D
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very low activity
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
medicine
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