Savle P S, Shelton T E, Meadows C A, Potts M, Gandour R D, Kennelly P J
Department of Chemistry, Institute for Genomics, Blacksburg, Virginia 24061, USA.
Arch Biochem Biophys. 2000 Apr 15;376(2):439-48. doi: 10.1006/abbi.2000.1750.
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 beta-naphthyl phosphate represented an excellent general substrate for both PTPs and DSPs. While DSPs tended to hydrolyze alpha-naphthyl phosphate at rates comparable to that of the beta-isomer, the PTPs PTP-1B and Tc-PTPa did not. PTP-H1, however, displayed high alpha-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 >/=10(4)-10(6). 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 beta-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.
三种双特异性磷酸酶（DSPs），即IphP、VHR和Cdc14，以及三种蛋白酪氨酸磷酸酶（PTPs），即PTP-1B、PTP-H1和Tc-PTPa，用一组低分子量磷酸酯进行了测试，以探究这两个在机制上同源的蛋白磷酸酶家族所表现出的不同底物特异性背后的因素。观察到磷酸β-萘酯是PTPs和DSPs的一种优异的通用底物。虽然DSPs倾向于以与β-异构体相当的速率水解磷酸α-萘酯，但PTPs中的PTP-1B和Tc-PTPa则不然。然而，PTP-H1表现出较高的磷酸α-萘酯酶活性。有趣的是，与PTP-1B或Tc-PTPa相比，PTP-H1在体外还表现出高得多的蛋白丝氨酸磷酸酶活性，相对于等效酪氨酸磷酸化蛋白，其活性为0.2 - 0.3%。后两种PTPs区分两种测试蛋白的丝氨酸磷酸化形式和酪氨酸磷酸化形式的系数≥10⁴ - 10⁶。虽然游离磷酸丝氨酸是所有测试的DSPs的极差底物，但添加一个疏水“柄”形成N-（环己烷甲酰基）-O-磷酸-L-丝氨酸后产生了一种化合物，IphP能高效水解该化合物，即水解速率与游离磷酸酪氨酸或对硝基苯磷酸相当。VHR也能以约为磷酸β-萘酯十分之一的速率水解N-（环己烷甲酰基）-O-磷酸-L-丝氨酸（1 mM）。所测试的PTPs均未表现出针对该化合物的显著活性。然而，N-（环己烷甲酰基）-O-磷酸-L-丝氨酸并非DSPs的通用底物，因为Cdc14几乎没有水解它的倾向。
