Roles of Gln81 and Cys80 in catalysis by glycosylphosphatidylinositol-phospholipase C from Trypanosoma brucei.
Roles of Gln81 and Cys80 in catalysis by glycosylphosphatidylinositol-phospholipase C from Trypanosoma brucei.
复制标题
Gln81 和 Cys80 在布氏锥虫糖基磷脂酰肌醇磷脂酶 C 催化中的作用。
DOI：
10.1046/j.1432-1327.1999.00690.x
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Mensa-Wilmot,K
中科院分区：
文献类型：
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作者：
Rashid,MB;Russell,M;Mensa-Wilmot,K
Glycosylphosphatidylinositol‐specific phospholipase C (GPtdIns‐PLC) is found in the protozoan parasiteTrypanosoma brucei. A region of protein sequence similarity exists between the protozoan enzyme and eubacterial phosphatidylinositol‐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, andkcatwas reduced 50‐fold. Thus an amide side‐chain at residue 81 is essential for catalysis by GPtdIns‐PLC.Sulfhydryl reagents inactivate GPtdIns‐PLC, suggesting that a Cys could be close to the enzyme active site. Surprisingly,p‐chloromercuriphenyl sulfonate (p‐CMPS) is significantly more potent thanN‐ethylmaleimide, the less bulky compound. This knowledge prompted us to test whether replacement of Cys80 with an amino acid possessing a bulky side‐chain would inactivate GPtdIns‐PLC: Cys80Ala, Cys80Thr, Cys80Phe, Cys184Ala, and Cys269‐270‐273Ser were constructed for that purpose. Cys80Phe lacked enzyme activity, while Cys80Ala, Cys80Thr and Cys269‐270‐273Ser retained 33 to 100% of wild‐type activity. 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, a bulky adduct that inactivated GPtdIns‐PLC, similar to the Cys80Phe mutation. We conclude that a bulky side‐chain (or adduct) at position 80 of GPtdIns‐PLC abolishes enzyme activity. Together, these observations place Cys80 and Gln81 at, or close to, the active site of GPtdIns‐PLC fromT. brucei.
