N-(cyclohexanecarboxyl)-O-phospho-l-serine, a minimal substrate for the dual-specificity protein phosphatase IphP.
N-(cyclohexanecarboxyl)-O-phospho-l-serine, a minimal substrate for the dual-specificity protein phosphatase IphP.
复制标题
N-(环己烷羧基)-O-磷酸-L-丝氨酸，双特异性蛋白磷酸酶 IphP 的最小底物。
DOI：
10.1006/abbi.2000.1750
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发表时间：
2000
影响因子：
3.9
通讯作者：
Kennelly,PJ
中科院分区：
文献类型：
--
作者：
Savle,PS;Shelton,TE;Meadows,CA;Potts,M;Gandour,RD;Kennelly,PJ
三个双特异性磷酸酶[DSP]，IphP，VHR和Cdc 14，和三个蛋白质酪氨酸磷酸酶[PTP]，PTP-1B，PTP-H1和Tc-PTPa，挑战与一组低分子量的磷酸酯，以探测的因素，由这两个机制同源的蛋白质磷酸酶家族显示不同的底物特异性。据观察，β-磷酸萘酯代表了PTP和DSP的优异的通用底物。虽然DSP倾向于以与β-异构体相当的速率水解α-磷酸萘酯，但PTP PTP-1B和Tc-PTPa则不然。而PTP-H1则表现出较高的α-萘基磷酸酶活性。有趣的是，PTP-H1在体外也显示出比PTP-1B或Tc-PTPa高得多的蛋白丝氨酸磷酸酶活性，相当于酪氨酸磷酸化蛋白的0.2-0.3%。后两种PTP通过≥104-106的因子区分两种供试蛋白的丝氨酸和酪氨酸磷酸化形式。虽然游离的磷酸丝氨酸代表了对于所有检测的DSP而言非常差的底物，但是添加疏水性“柄”以形成N-（环己烷羧基）-O-磷酸-1-丝氨酸产生了被IphP高效水解的化合物，即，其速率与游离磷酸酪氨酸或磷酸对硝基苯酯的速率相当。VHR还水解N-（环己烷羧基）-O-磷酸-L -丝氨酸（1 mM），水解速率约为磷酸萘酯的十分之一。测试的PTP均未表现出对该化合物的显著活性。然而，N-（环己烷羧基）-O-磷酸-L -丝氨酸并没有被证明是DSP的通用底物，因为Cdc 14几乎没有显示出水解它的倾向。
Three dual-specific phosphatases [DSPs], IphP, VHR, and Cdc14, and three protein-tyrosine phosphatases [PTPs], PTP-1B, PTP-H1, and Tc-PTPa, were challenged with a set of low molecular weight phosphoesters to probe the factors underlying the distinct substrate specificities displayed by these two mechanistically homologous families of protein phosphatases. It was observed that β-naphthyl phosphate represented an excellent general substrate for both PTPs and DSPs. While DSPs tended to hydrolyze α-naphthyl phosphate at rates comparable to that of the β-isomer, the PTPs PTP-1B and Tc-PTPa did not. PTP-H1, however, displayed high α-naphthyl phosphatase activity. Intriguingly, PTP-H1 also displayed much higher protein-serine phosphatase activity in vitro, 0.2–0.3% that toward equivalent tyrosine phosphorylated proteins, than did PTP-1B or Tc-PTPa. The latter two PTPs discriminated between the serine- and tyrosine-phosphorylated forms of two test proteins by factors of ≥104–106. While free phosphoserine represented an extremely poor substrate for all of the DSPs examined, the addition of a hydrophobic “handle” to form N-(cyclohexanecarboxyl)-O-phospho-l -serine produced a compound that was hydrolyzed by IphP with high efficiency, i.e., at a rate comparable to that of free phosphotyrosine or p-nitrophenyl phosphate. VHR also hydrolyzed N-(cyclohexanecarboxyl)-O-phospho-l -serine (1 mM) at a rate approximately one-tenth that of β-naphthyl phosphate. None of the PTPs tested exhibited significant activity against this compound. However, N-(cyclohexanecarboxyl)-O-phospho-l -serine did not prove to be a universal substrate for DSPs as Cdc14 displayed little propensity to hydrolyze it.