H-Ras peptide and protein substrates bind protein farnesyltransferase as an ionized thiolate
H-Ras peptide and protein substrates bind protein farnesyltransferase as an ionized thiolate
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DOI：
10.1021/bi981525v
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发表时间：
1998-11-03
期刊：
影响因子：
2.9
通讯作者：
Fierke, CA
中科院分区：
文献类型：
--
作者：
Hightower, KE;Huang, CC;Fierke, CA
锌金属酶蛋白法尼基转移酶（FTase）催化蛋白质底物的半胱氨酸残基与15个碳的法尼基基团的烷基化。我们已经开发了荧光测定法来直接测量酶对肽和蛋白质（Ras）底物的亲和力。对应于H-Ras的羧基末端的肽在生理pH下在μ M范围内（K-D = 4 μ M）与FT酶结合;然而，肽亲和力在与酶结合的法呢基二磷酸（FPP）类似物的三元复合物中增强约70倍，表明两种底物协同结合。还研究了底物结合的pH依赖性，并观察到两种电离：对于三元复合物，pK（a）值为8.1，反映游离肽的巯基电离，和6.4。Co2+取代的FTase三元复合物的光学吸收光谱中的配体-金属电荷转移带的pH依赖性表明，金属配位的硫醇离子化，pK（a）为6.3。这些数据表明，肽硫与FTase中锌离子的金属配位降低了硫醇的pK（a），导致在生理pH下形成结合的硫醇盐。
The zinc metalloenzyme protein farnesyltransferase (FTase) catalyzes the alkylation of a cysteine residue of protein substrates with a 15 carbon farnesyl group. We have developed fluorescence assays to directly measure the affinity of the enzyme for peptide and protein (Ras) substrates. A peptide corresponding to the carboxyl terminus of H-Ras binds to FTase in the mu M range (K-D = 4 mu M) at physiological pH; however, the peptide affinity is enhanced approximately 70-fold in a ternary complex with an enzyme-bound farnesyl diphosphate (FPP) analogue, indicating that the two substrates bind synergistically. The pH dependence of substrate binding was also investigated, and two ionizations were observed: for the ternary complex, the pK(a) values are 8.1, reflecting ionization of the thiol of the free peptide, and 6.4. The pH dependence of the ligand-metal charge-transfer band in the optical absorption spectra of a Co2+-substituted FTase ternary complex suggests that a metal-coordinated thiol ionizes with a pK(a) of 6.3. These data indicate that metal coordination of the peptide sulfur with the zinc ion in FTase lowers the pK(a) of the thiol resulting in formation of a bound thiolate at physiological pH.