Information on EC 3.4.21.46 - complement factor D and Organism(s) Homo sapiens

for references in articles please use BRENDA:EC3.4.21.46
Word Map on EC 3.4.21.46
Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
Specify your search results
Select one or more organisms in this record:
This record set is specific for:
Homo sapiens


The taxonomic range for the selected organisms is: Homo sapiens

The enzyme appears in selected viruses and cellular organisms

top print hide
EC NUMBER
COMMENTARY hide
3.4.21.46
-
top print hide
RECOMMENDED NAME
GeneOntology No.
complement factor D
top
top print hide
CAS REGISTRY NUMBER
COMMENTARY hide
37213-56-2
-
top print hide
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
top print hide
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
additional information
-
the substrate-binding, or exosite, region displays a well defined and rigid conformation. A salt bridge between Arg202 on the self-inhibitory loop and Asp177 locks the S1 pocket and rigidifies the exosite. Asp-His-Ser catalytic site. The enzyme exhibits conformational dynamics like thrombin, but unlike in thrombin a mechanism has evolved in factor D that locks the unbound native state into an ordered inactive conformation via the self-inhibitory loop. The Asp-His-Ser catalytic site of the enzyme is in an enzymatically active conformation induced by substrate binding at the exosite
top print hide
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
-
-
-
-
?
factor B + H2O
Ba + Bb
show the reaction diagram
-
-
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
-
-
-
-
?
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
-
-
-
-
?
pro-convertase C3bB + H2O
active C3 convertase C3bBb + ?
show the reaction diagram
-
-
-
-
?
additional information
?
-
top print hide
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
-
-
-
-
?
additional information
?
-
top print hide
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
-
required
top print hide
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3,4-dichloroisocoumarin
-
mechanism-based inactivation
anti-factor D Fab fragment
-
an Escherichia coli-expressed, humanized Fab fragment, derived from monoclonal antibody 166-32, and designed as specific enzyme inhibitor targeting the exosite. Anti-factor D Fab fragment prevents factor D-mediated proteolytic activation of its macromolecular substrate C3bB, but not proteolysis of a small synthetic substrate, indicating that the inhibitor does not block access of the substrate to the catalytic site, binding structure and inhibitory mechanism, overview. The structures show that the AFD-binding site includes surface loops of the enzyme that form part of the enzyme's exosite. Anti-factor D Fab fragment inhibits the enzyme proteolytic function by interfering with macromolecular substrate access rather than by inhibiting the enzyme catalysis
-
diisopropyl fluorophosphate
-
-
isatoic anhydride
-
-
additional information
-
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
-
top print hide
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
complement factor B
-
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
-
additional information
-
top print hide
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.95 - 11.44
Benzyloxycarbonyl-Lys-thiobenzyl ester
top print hide
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.3 - 21.3
Benzyloxycarbonyl-Lys-thiobenzyl ester
top print hide
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
top print hide
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.1 - 7.2
-
-
top print hide
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
low levels of vegf-D mRNA
Manually annotated by BRENDA team
-
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
parasitized red blood cells, pRBC, infected by Plasmodium falciparum, rosetting serum dependent for the strains FCR3S1.2 and Malayan Camp
Manually annotated by BRENDA team
-
PDGF-D high-expressing breast cancer cell line SUM-149 is more invasive compared to low-expressing cell lines
Manually annotated by BRENDA team
-
normal hepatocytes synthesize factor D constitutively. The liver may be a major source of plasma factor D
Manually annotated by BRENDA team
-
low levels of vegf-D mRNA
Manually annotated by BRENDA team
-
PDGF-D is uniformly expressed in visceral epithelial cells and arteriolar wall in normal kidneys, kidneys with acute vascular rejection, and kidneys with chronic allograft nephropathy. In kidneys with acute vascular rejection, PDGF-D is expressed in arterial medial smooth muscle cells, some adventitial cells, and some neointimal cells that also have alpha-smooth muscle actin expression
Manually annotated by BRENDA team
-
low PDGF-D expressing breast cancer cell line is less invasive
Manually annotated by BRENDA team
-
PDGF-D expressing breast cancer cell lines are more invasive compared to non-expressing cell lines
Manually annotated by BRENDA team
-
low PDGF-D expressing breast cancer cell line is less invasive
Manually annotated by BRENDA team
-
low levels of vegf-D mRNA
Manually annotated by BRENDA team
-
5- to 8fold higher amounts of vegf-D mRNA as the other gastric cell lines
Manually annotated by BRENDA team
additional information
top print hide
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
-
occasionally in peritumoral stroma
-
Manually annotated by BRENDA team
top print hide
PDB
SCOP
CATH
UNIPROT
ORGANISM
Homo sapiens;
top print hide
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
21000
-
Westernblot
22900
-
equilibrium sedimentation
23000
-
x * 23000, SDS-PAGE
24000
-
x * 24000, SDS-PAGE
24376
-
x * 24376, calculation from nucleotide sequence
25000
-
x * 25000, SDS-PAGE
top print hide
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
-
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
top print hide
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
no modification
-
no potential N-glycosylation site, no carbohydrate is present in the purified protein
proteolytic modification
-
profactor D
top print hide
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure of profactor D
-
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
-
S183A mutant protein in complex with pro-convertase C3bB
-
structure of diisopropyl fluorophosphate-inhibited factor D
-
structure of native factor D and a complex formed with isatoic anhydride inhibitor
-
wild type and mutant factor D, expression in CHO cells
-
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
-
top print hide
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
freezing and thawing causes approximately 30% loss of activity
-
top print hide
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C, no aggregation occurs, stable
-
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
-
top print hide
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cation-exchange chromatography,gelfiltration
-
enzyme and inhibitor by gel filtration
-
recombinant soluble wild-type and mutant enzymes from HEK-293-E cells by cation exchange chromatography and gel filtration
-
Sephacryl S-100 and DEAE Sepharose fast flow column chromatography, 4°C
-
VEGF-DDELTANDELTAC and the three cysteine mutants purified by metal affinity chromatography
-
top print hide
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
-
expressed in breast cancer cell lines MDA-MB-468 and MCF-7 cells
-
expressed in HEK293-E cells
-
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
-
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
-
transient expression of wild-type and mutant enzymes in HEK-293-E cells from pUPE.05.05 expression vectors, the recombinant proteins are secreted
-
top print hide
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
-
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
-
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
-
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
-
top print hide
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C25A
-
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
-
has biological activity comparable to that of the native protein in Ba/F3-VEGFR-2 cells
C25I
-
is mainly in a presumably covalently bound dimeric form
C25L
-
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
-
is mainly in a presumably covalently bound dimeric form
C44A
-
does not bind to the soluble receptor VEGFR-2/IgGFc
C53A
-
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
-
catalytically inactive
P43S
-
can not induce cell survival
R115C
-
activity like wild type
R157A
-
catalytically inactive
R22I
-
can not induce cell survival
R22I/C25L
-
has biological activity comparable to that of the native protein in Ba/F3-VEGFR-2 cells, has increased dimer to monomer ratio
R22L
-
can not induce cell survival
R22L/C25L
-
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
-
site-directed mutagenesis, the mutation causes steric hindrance of Trp199 resulting in pronounced rearrangement of the proteinase active-site region, structure analysis
S94Y
-
6fold increase in turnover number
S94Y/T214/S215W
-
the triple mutant has a turnover number that is 16fold higher than that of wild type factor D
T214S/S215W
-
more than 16fold incerease of the ratio of turnover number/KM-value
T638G
-
factor D deficient mutant
T640C
-
factor D deficient mutant
V203D
-
very low activity
top print hide
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
-
the enzyme's exosite is a target for design of specific inhibitors for inhibition of the pathway complement activation in advanced dry age-related macular degeneration
medicine
top
top