Abstract
The glycosylphosphatidylinositol phospholipase C (GPI-PLC) from Trypanosoma brucei is particularly effective in hydrolysing the GPI-anchors of some proteins. The enzyme is inhibited by Zn2+ and p-chloromercurylphenylsulphonic acid, both of which can act as sulphydryl reagents, suggesting that a cysteine residue may be important in catalysis. Single cysteine to serine mutants have been produced for all eight cysteines in GPI-PLC; all the mutants were fully active in vitro and were still susceptible to p-chloromercurylphenylsulphonic acid inhibition. In contrast, a single histidine 34 to glutamine mutation totally inactivated GPI-PLC. The histidine was chosen after a sequence alignment with the Bacillus cereus phosphatidylinositol phospholipase C (PI-PLC) suggested a conservation of active site residues, including histidine 34 which is central to the proposed reaction mechanism (Heinz D.W., Ryan M., Bullock T.L., Griffith O.H. EMBO J 1995;14:3855-3863). The results suggest that the GPI-PLC and bacterial PI-PLCs have conserved active sites and that the inhibition of GPI-PLC by sulphydryl reagents can occur through more than one residue.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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4-Chloromercuribenzenesulfonate / pharmacology
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Amino Acid Sequence
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Animals
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Bacillus cereus / enzymology
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Bacillus cereus / genetics
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Binding Sites
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Cysteine / chemistry
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Electrophoresis, Polyacrylamide Gel
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Enzyme Inhibitors / pharmacology
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Escherichia coli / genetics
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Glycosylphosphatidylinositol Diacylglycerol-Lyase
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Histidine / chemistry
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Listeria monocytogenes / enzymology
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Listeria monocytogenes / genetics
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Phosphatidylinositol Diacylglycerol-Lyase
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Phosphoinositide Phospholipase C
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Recombinant Proteins / metabolism
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Sequence Alignment
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Sulfhydryl Reagents / pharmacology
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Trypanosoma brucei brucei / enzymology*
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Trypanosoma brucei brucei / genetics
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Type C Phospholipases / antagonists & inhibitors
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Type C Phospholipases / chemistry
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Type C Phospholipases / genetics
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Type C Phospholipases / metabolism*
Substances
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Enzyme Inhibitors
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Recombinant Proteins
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Sulfhydryl Reagents
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Histidine
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4-Chloromercuribenzenesulfonate
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Type C Phospholipases
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Phosphoinositide Phospholipase C
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Phosphatidylinositol Diacylglycerol-Lyase
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Glycosylphosphatidylinositol Diacylglycerol-Lyase
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Cysteine