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 Zn 2+ 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.
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