SYNTHESIS OF PHOSPHOTYROSINE-CONTAINING PEPTIDES AND THEIR USE AS SUBSTRATES FOR PROTEIN-TYROSINE PHOSPHATASES
SYNTHESIS OF PHOSPHOTYROSINE-CONTAINING PEPTIDES AND THEIR USE AS SUBSTRATES FOR PROTEIN-TYROSINE PHOSPHATASES
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DOI：
10.1021/bi00067a027
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发表时间：
1993-04-27
期刊：
影响因子：
2.9
通讯作者：
BARANY, G
中科院分区：
文献类型：
--
作者：
OTTINGER, EA;SHEKELS, LL;BARANY, G
用于化学合成含磷酸酪氨酸的肽的现有方法涉及在肽组装完成后将完全保护的磷酸氨基酸掺入到肽链中或游离酚侧链的磷酸化。本工作描述了一种新的和一般的方法，用于固相合成磷酸肽，具有直接掺入N（α）-（9-芴甲氧羰基）-O-磷酸-L-酪氨酸（未保护的侧链）。该技术避免了含有酪氨酸H-膦酸酯的肽副产物的形成，这是文献亚磷酸化/氧化方法中以前未认识到的副反应。通过较新的优选方法合成与脂肪细胞脂质结合蛋白的酪氨酸磷酸化位点相对应的磷酸肽。通过高效液相色谱（HPLC）、毛细管区带电泳（CZE）、氨基酸分析（AAA）、快原子轰击质谱（FABMS）和P-31核磁共振（P-31 NMR）对这些肽进行纯化和表征。合成的肽被测试为两种不同的蛋白酪氨酸磷酸酶，大鼠脑蛋白酪氨酸磷酸酶（PTP 3）和人酸性磷酸酶的底物。底物特异性在pH 6.0和37 ℃下测定，使用比色法测定释放的无机磷酸盐。动力学分析表明，大鼠脑PTK和人脂肪细胞酸性磷酸酶催化肽脱磷酸化，但具有不同的速率和亲和力。大鼠脑PTODN显示经典的Michaelis-Menten动力学，对于长度为4和10个残基的磷酸化肽，K（m）分别为68 +/- 9 μ M和42 +/- 11 μ M，k（cat）/K（m）值分别为4.9 × 10（5）s-1 M-1和6.9 × 10（5）s-1 M-1。相比之下，人酸性磷酸酶表现出线性动力学，没有饱和观察到高达2 mM的磷酸肽。这些结果证明了该方法的实用性，并描述了如何合成酪氨酸磷酸肽可用于评估水解蛋白磷酸酪氨酸的酶的催化功效。
Prior methods for the chemical synthesis of phosphotyrosine-containing peptides involved the incorporation of fully protected phosphoamino acids into the peptide chain or phosphorylation of free phenol side chains after peptide assembly is complete. The present work describes a novel and general methodology for the solid-phase synthesis of phosphopeptides, featuring direct incorporation of N(alpha)-(9-fluorenylmethyloxycarbonyl)-O-phospho-L-tyrosine (unprotected side chain). This technique obviated the formation of peptide byproducts containing tyrosine H-phosphonate, a previously unrecognized side reaction from literature phosphitylation/oxidation approaches. Phosphopeptides corresponding to the tyrosine phosphorylation site of adipocyte lipid binding protein were synthesized by the newer, preferred method. These peptides were purified and characterized by high-performance liquid chromatography (HPLC), capillary zone electrophoresis (CZE), amino acid analysis (AAA), fast atom bombardment mass spectrometry (FABMS), and P-31 nuclear magnetic resonance (P-31 NMR). The synthetic peptides were tested as substrates for two distinct protein tyrosine phosphatases, rat brain protein tyrosine phosphatase (PTPase) and human acid phosphatase. Substrate specificity was measured at pH 6.0 and 37-degrees-C, using a colorimetric assay for released inorganic phosphate. Kinetic analysis revealed that both the rat brain PTPase and the human adipocyte acid phosphatase catalyzed peptide dephosphorylation but with different rates and affinities. The rat brain PTPase displayed classical Michaelis-Menten kinetics, with K(m)'s of 68 +/- 9 muM and 42 +/- 11 muM and k(cat)/K(m) values of 4.9 X 10(5) s-1 M-1 and 6.9 x 10(5) s-1 M-1 determined for phosphorylated peptides of lengths 4 and 10 residues, respectively. In contrast, the human acid phosphatase demonstrated linear kinetics, with no saturation observed up to 2 mM phosphopeptide. These results demonstrate the utility of the methodology and describe how synthetic tyrosine phosphopeptides can be used to assess the catalytic efficacies of enzymes that hydrolyze protein phosphotyrosine.