Cysteine-less glycosylphosphatidylinositol-specific phospholipase C is inhibited competitively by a thiol reagent: evidence for glyco-mimicry by p-chloromercuriphenylsulphonate.
Cysteine-less glycosylphosphatidylinositol-specific phospholipase C is inhibited competitively by a thiol reagent: evidence for glyco-mimicry by p-chloromercuriphenylsulphonate.
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硫醇试剂竞争性抑制无半胱氨酸的糖基磷脂酰肌醇特异性磷脂酶 C：对氯汞苯磺酸盐模拟糖的证据。
DOI：
10.1042/bj20020367
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
Mensa-Wilmot,Kojo
中科院分区：
文献类型：
--
作者：
Stanton,JulieD;Rashid,MohammadB;Mensa-Wilmot,Kojo
Glycosylphosphatidylinositol (GPI)-specific phospholipases are highly valuable for studying the structure and function of GPIs. GPI-specific phospholipase C (GPI-PLC) fromTrypanosoma bruceiand phosphatidylinositol-specific phospholipase C (PI-PLC) fromBacillus cereusare the most widely studied of this class of phospholipases C. Inhibition of protein activity by thiol reagents is indicative of the participation of cysteine residues in biochemical events. The thiol reagentp-chloromercuriphenylsulphonate (pCMPS) inhibitsT. bruceiGPI-PLC, which has eight cysteine residues. Surprisingly, we found that the activity ofB. cereusPI-PLC is also blocked bypCMPS, although the protein does not contain cysteine residues. Inhibition ofB. cereusPI-PLC was reversed whenpCMPS was size-separated from a preformedpCMPS·PI-PLC complex. In contrast, no activity was recovered whenT. bruceiGPI-PLC was subjected to a similar protocol. Equimolar β-mercaptoethanol (β-ME) reversed the inhibition of PI-PLC activity in a pCMPS·PI-PLC complex. ForT. bruceiGPI-PLC, however, ultrafiltration of the pCMPS·GI-PLC complex and addition of a large excess of β-ME was necessary for partial recovery of enzyme activity. ThusT. bruceiGPI-PLC is susceptible to inactivation by covalent modification withpCMPS, whereas PI-PLC is not. Kinetic analysis indicated thatpCMPS was a competitive inhibitor of PI-PLC when a GPI was a substrate. Curiously, with phosphatidylinositol as substrate, inhibition was no longer competitive. These data suggest thatpCMPS is a glyco-mimetic that occupies the glycan binding site of PI-PLC, from where, depending on the substrate, it inhibits catalysis allosterically or competitively.
