Roles of Gln81 and Cys80 in catalys is by glycosylphosphatidylinositol-phospholipase C from Trypanosoma brucei

Glycosylphosphatidylinositol-specific phospholipase C (GPtdIns-PLC) is foun d in the protozoan parasite Trypanosoma brucei. A region of protein sequenc e similarity exists between the protozoan enzyme and eubacterial phosphatid ylinositol-phospholipases C. The functional relevance of Cys80 and Gln81 of GPtdIns-PLC, both in this region, was tested with a panel of mutations at each position. Gln81Glu, Gln81Ala, Gln81Gly, Gln81Lys and Gln81Leu mutants were inactive. Cleavage of GPtdIns was detectable in Gln81Asn, although the specific activity decreased 500-fold, and k(cat) was reduced 50-fold. Thus an amide side-chain at residue 81 is essential for catalysis by GPtdIns-PL C. Sulfhydryl reagents inactivate GPtdIns-PLC, suggesting that a Cys could be close to the enzyme active site. Surprisingly, p-chloromercuriphenyl sulfon ate (p-CMPS) is significantly more potent than N-ethylmaleimide, the less b ulky compound. This knowledge prompted us to test whether replacement of Cy s80 with an amino acid possessing a bulky side-chain would inactivate GPtdI ns-PLC: Cys80Ala, Cys80Thr, Cys80Phe, Cys184Ala, and Cys269-270-273Ser were constructed for that purpose. Cys80Phe lacked enzyme activity, while Cys80 Ala, Cys80Thr and Cys269-270-273Ser retained 33 to 100% of wild-type activi ty. Interestingly, the Cys80Ala and Cys80Thr mutants became resistant to p- CMPS, as predicted if the sulfhydryl reagent reacted with Cys80 in the wild -type enzyme to form a cysteinyl mercurylphenylsulfonate moiety, ii bulky a dduct that inactivated GPtdIns-PLC, similar to the Cys80Phe mutation. We co nclude that a bulky side-chain (or adduct) at position XO of GPtdIns-PLC ab olishes enzyme activity. Together, these observations place Cys80 and Gln81 at, or close to, the active site of GPtdIns-PLC from T. brucei.