Information on EC 4.6.1.14 - glycosylphosphatidylinositol diacylglycerol-lyase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
4.6.1.14
-
RECOMMENDED NAME
GeneOntology No.
glycosylphosphatidylinositol diacylglycerol-lyase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
6-(alpha-D-glucosaminyl)-1-phosphatidyl-1D-myo-inositol = 6-(alpha-D-glucosaminyl)-1D-myo-inositol 1,2-cyclic phosphate + 1,2-diacyl-sn-glycerol
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
P-O bond cleavage
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
6-(alpha-D-glucosaminyl)-1-phosphatidyl-1D-myo-inositol 1,2-diacyl-sn-glycerol-lyase [6-(alpha-D-glucosaminyl)-1D-myo-inositol 1,2-cyclic phosphate-forming]
This enzyme is also active when O-4 of the glucosamine is substituted by carrying the oligosaccharide that can link a protein to the structure. It therefore cleaves proteins from the lipid part of the glycosylphostphatidylinositol (GPI) anchors. In some cases, the long-chain acyl group at the sn-1 position of glycerol is replaced by an alkyl or alk-1-enyl group. In other cases, the diacylglycerol is replaced by ceramide (see Lip-1.4 and Lip-1.5 for definition). The only characterized enzyme with this specificity is from Trypanosoma brucei, where the acyl groups are myristoyl, but the function of the trypanosome enzyme is unknown. Substitution on O-2 of the inositol blocks action of this enzyme. It is not identical with EC 3.1.4.50, glycosylphosphatidylinositol phospholipase D.
CAS REGISTRY NUMBER
COMMENTARY hide
129070-68-4
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
peanut
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
-
loss of GPI-phospholipase C polypeptide is associated with sustained replication of nascent procyclics
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1,2-dimyristoyl-sn-phosphatidylinositol + H2O
?
show the reaction diagram
-
poor substrate
-
-
?
6-(alpha-D-glucosaminyl)-1-phosphatidyl-1D-myo-inositol
6-(alpha-D-glucosaminyl)-1D-myo-inositol 1,2-cyclic phosphate + 1,2-diacyl-sn-glycerol
show the reaction diagram
acetylcholinesterase
soluble acetylcholinesterase + 1,2-diacylglycerol
show the reaction diagram
dimyristyl-P + H2O
?
show the reaction diagram
-
-
-
-
?
glycolipid A
?
show the reaction diagram
glycolipid Y
?
show the reaction diagram
glycosylated phosphatidylinositol + H2O
?
show the reaction diagram
-
-
-
-
?
glycosylphosphatidylinositol-anchored protein
?
show the reaction diagram
-
-
-
-
?
lipid A
?
show the reaction diagram
-
biological precursor of variant-surface-glycoprotein glycolipid
-
-
?
membrane form variant surface glycoprotein + H2O
soluble variant surface glycoprotein + sn-1,2-dimyristoylglycerol
show the reaction diagram
-
mfVSG, enzyme catalyzes the conversion of membrane form variant surface glycoproteins to soluble variant surface glycoproteins, with the release of sn-1,2-dimyristylglycerol
-
-
?
phosphatidylinositol
inositol 1,2-cyclic phosphate + inositol phosphate
show the reaction diagram
pronase fragment + H2O
?
show the reaction diagram
-
aspartylethanolamine-glycolipid moiety of mfVSG
-
-
?
pronase fragment of mfVSG + H2O
?
show the reaction diagram
-
contains only the terminal amino acid and the glycolipid anchor
-
-
?
variant surface glycoprotein
?
show the reaction diagram
variant-surface-glycoprotein
1,2-didecanoylglycerol + soluble variant-surface-glycoprotein
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
6-(alpha-D-glucosaminyl)-1-phosphatidyl-1D-myo-inositol
6-(alpha-D-glucosaminyl)-1D-myo-inositol 1,2-cyclic phosphate + 1,2-diacyl-sn-glycerol
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Hg2+
-
10 microM 100% inhibition, 1 microM 67% activity left
Zn2+
-
100 microM 100% inhibition, 10 microM 59% activity left
additional information
-
calcium-independent
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-deoxy-2-fluoro-scyllo-inositol-1-O-dodecyl-phosphonic acid
Cetrimide
-
i.e. alkyltrimethylammonium bromide, 0.3 mg/ml, neutral enzyme form
deoxycholate
-
neutral enzyme form, above 1 mg/ml, activation at 0.5-1 mg/ml
Glucosaminyl-alpha-1,6-2-deoxy-D-myo-inositol
-
i.e. compound VP-615L, more effective than VP-606L
Glucosaminyl-alpha-1,6-D-myo-inositol
-
i.e. compound VP-606L or 6-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-D-myo-inositol
Glucosaminyl-alpha-1,6-D-myo-inositol 1,2-cyclic phosphate
-
i.e. compound VP-601L, product inhibition
Glucosaminyl-alpha-1,6-D-myo-inositol 1-dodecylphosphonate
-
i.e. compound VP-604L
Glucosaminyl-alpha-1,6-D-myo-inositol 1-hexylphosphonate
-
i.e. compound VFT-2
glucosaminyl-alpha-1,6-D-myo-inositol 1-phosphate
-
i.e. compound VP-600L
Hg2+
-
10 microM 100% inhibition, 1 microM 67% activity left
Inositol 1-dodecylphosphonate
-
i.e. compound VP-602L
KCl
-
0.125 M
Mannosyl-alpha-1,4-glucosaminyl-alpha-1,6-D-myo-inositol
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i.e. O-alpha-D-mannopyranosyl-1,4-O-2-amino-2-deoxy-alpha-D-glucopyranosyl-1,6-D-myo-inositol
Mg2+
-
weak, 5 mM
myo-inositol-1,2-cyclo-dodecyl-phosphonic acid
myo-inositol-1-O-dodecylphosphonic acid methylester
-
GPI-2349
N-(N,N-Dimethylcarbamyl)-glucosaminyl-alpha-1,6-D-myo-inositol
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i.e. compound VC-109B, less effective than VP-606L
N-Acetylglucosaminyl-alpha-1,6-D-myo-inositol
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i.e. compound VC-105B, weak
NaCl
-
0.125 M
NH4Cl
-
0.125 M
Nonidet P-40
-
above 0.1% w/v, neutral enzyme form
p-Chloromercuriphenylsulfonic acid
phosphatidylcholine
-
weak
phosphatidylglycerol
-
-
phosphatidylinositol
-
-
phosphatidylserine
-
-
propanolol
-
-
sulfhydryl reagents
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-
Triton X-100
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0.05-0.5%, activation at 0.02%
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
butanol
-
activation, 2% v/v, acidic enzyme form, not neutral enzyme form
deoxycholate
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activation, 0.5-1 mg/ml, neutral enzyme form, not acidic enzyme form, inhibits above 1 mg/ml
dithiothreitol
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enzyme activity shows marked stimulation in the presence of the thiol-reducing agent, DTT
DTT
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activation, 0.025 M
EDTA
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activation, 5 mM
glibenclamide
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-
glimepiride
-
-
glimepiride M1
-
-
-
palmitate
-
-
Triton X-100
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activation, above critical micelle concentration, inhibition from 0.05% to 0.5%
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0008
Acetylcholinesterase
-
-
-
0.0003 - 0.0021
myristate-labeled variant-surface-glycoprotein
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0.037
phosphatidylinositol
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0.0004 - 0.0027
Variant-surface-glycoprotein
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.65 - 27.3
Variant-surface-glycoprotein
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0006
myo-inositol-1,2-cyclo-dodecyl-phosphonic acid
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-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00245
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last purification step, PI-Sepharose
1.59
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Mono P fraction
280000
-
C80A
1100000
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wild-type
3500000
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C80T
11000000
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concentrated CM-Sephadex pool
additional information
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in high cholesterol-containing detergent-soluble glycolipid-enriched membrane microdomains: 488% of the activity measured in plasma membranes, in low cholesterol-containing detergent-soluble glycolipid-enriched membrane microdomains: 141% of the activity measured in plasma membranes, in non-detergent-soluble glycolipid-enriched membrane microdomains: 29% of the activity measured in plasma membranes
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 5
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acidic enzyme form
5.5 - 6
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-
6.5 - 7
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neutral enzyme form
7.5 - 8.5
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-
10
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two maxima, one at pH 7.0 and the other at pH 10.0, that shows the highest enzyme activity
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 9.5
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about half-maximal activity at pH 6.5 and 9.5, little or no activity below pH 6
7 - 10
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pH dependence of GPI-PLC activity. Two maxima, one at pH 7.0 and the other at pH 10.0, that shows the highest enzyme activity
7.4 - 9.1
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-
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
neutral enzyme form
Manually annotated by BRENDA team
additional information
-
bloodstream-form Trypanosoma brucei RUMP528 (GPI-PLC-/-)
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
neutral enzyme form
-
Manually annotated by BRENDA team
-
in case of mutations on the endosome targeting motif, glycosomal GPI-PLC from Trypanosoma brucei fails to produce the glycosylphosphatidylinositol deficiency in Leishmania major
Manually annotated by BRENDA team
-
predominantly acidic enzyme form
Manually annotated by BRENDA team
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37000
-
1 * 37000, SDS-PAGE
39000
-
PAGE
39800
-
SDS-PAGE, visualized either by Coomassie Blue or by silver staining
47000
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gel filtration, sedimentation equilibrium centrifugation, in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
-
alpha2, 2 * 39000, gel filtration, dominates in presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
monomer
tetramer
-
alph4, 4 * 39000, gel filtration, dominates in presence of Nonident P-40
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
side-chain modification
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
10 min, stable
50
-
10 min, inactivation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
GPI-PLC is very stable, maintaining full activity at 4°C for 1-2 months
-
the enzyme is not sensitive to trypsin digest in live cells
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C, in crude membranes, 6 months
-
-70°C, purified enzyme preparation, at least 2 months
-
4°C, 30% loss within 5 days, 10% loss of activity in the presence of 50% glycerol
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
anion exchange chromatography on DEAE-cellulose followed by chromatography on phosphatidylinositol-Sepharose
-
recombinant protein
-
solubilized by n-octyl glucoside
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
-
expression in Escherichia coli; expression in Xenopus oocytes
-
expression in Leishmania major
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expression of a teracycline-inducible GPIPLC-gene in conditional knock-out bloodstraem forms and in procyclic cell line
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expression of the PLC elimination construct in Trypanosoma brucei
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mutants subcloned into pLew82(DLUC) vector, Cys mutants stably expressed in strain RUMP528
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overexpression in Escherichia coli
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
during differentiation of bloodstream to insect stage (procyclic), translation GPI-phospholipase C polypeptide mRNA ceases within 8 h of initiating transformation. Reduction of GPI-PLCp in early-stage procyclics is linked to parasite replication
-
expression of GPI-phospholipase C polypeptide in the parasite is restricted to the bloodstream form. Nascent procyclics contain 400fold more GPI-phospholipase C polypeptide than established insect stage. GPI-PLCp is retained in strains that fail to replicate after differentiation of the bloodstream to the procyclic form
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C184S
-
fully active in vitro and still susceptible to p-chloromercuriphenylsulfonic acid
C24A
-
exhibits properties indistinguishable from the unmutated enzyme, in exiting glycosomes after exposure to pH 9.1 or hypotonic buffer
C24S
-
fully active in vitro and still susceptible to p-chloromercuriphenylsulfonic acid
C269-270-273S
-
33% activity of wild-type enzyme
C269C/C270S/C273S
-
exhibits activity in the range detected for the unmutated protein, localization in glycosome
C269S
-
fully active in vitro and still susceptible to p-chloromercuriphenylsulfonic acid
C269S/C270S
C269S/C270S/C273S
-
exhibits properties indistinguishable from the unmutated enzyme, in exiting glycosomes after exposure to pH 9.1 or hypotonic buffer
C269S/C273S
C270S
-
fully active in vitro and still susceptible to p-chloromercuriphenylsulfonic acid
C270S/C273S
C273S
-
fully active in vitro and still susceptible to p-chloromercuriphenylsulfonic acid
C332A
-
exhibits properties indistinguishable from the unmutated enzyme, in exiting glycosomes after exposure to pH 9.1 or hypotonic buffer
C332S
-
fully active in vitro and still susceptible to p-chloromercuriphenylsulfonic acid
C347S
-
fully active in vitro and still susceptible to p-chloromercuriphenylsulfonic acid
C80F
-
enzyme is inactive
C80S
-
fully active in vitro and still susceptible to p-chloromercuriphenylsulfonic acid
C80T
-
33% activity of wild-type enzyme, resistant to p-chloromercuriphenylsulfonic acid
H34Q
-
enzyme is totally inactive
Q81A
-
enzyme is inactive
Q81E
-
enzyme is inactive
Q81G
-
enzyme is inactive
Q81K
-
enzyme is inactive
Q81N
-
specific activity is 500fold decreased
C184A
-
exhibits properties indistinguishable from the unmutated enzyme, in exiting glycosomes after exposure to pH 9.1 or hypotonic buffer
-
C347A
-
can not exit glycosomes after treatment of cells expressing the protein with mild base or hypo-osmotic buffer
-
C80A
-
exhibits properties indistinguishable from the unmutated enzyme, in exiting glycosomes after exposure to pH 9.1 or hypotonic buffer
-
Q81L
-
fails to cleave glycosylphosphatidylinositols despite being transported from glycosomes to the endoplasmic reticulum after hypoosmotic or mild alkaline treatment of parasites, lacks enzyme activity
-
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine