Identification of a cysteine residue essential for activity of protein farnesyltransferase - Cys(299) is exposed only upon removal of zinc from the enzyme
Identification of a cysteine residue essential for activity of protein farnesyltransferase - Cys(299) is exposed only upon removal of zinc from the enzyme
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DOI：
10.1074/jbc.271.45.28541
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发表时间：
1996-11-08
影响因子：
4.8
通讯作者：
Casey, PJ
中科院分区：
文献类型：
--
作者：
Fu, HW;Moomaw, JF;Casey, PJ
蛋白质法尼基转移酶(FTase)是一种锌金属酶，它对许多蛋白质进行翻译后修饰，这对它们的功能至关重要。利用半胱氨酸特异性试剂研究了半胱氨酸残基在FT酶活性中的重要性。N-乙基马来酰亚胺(NEM)可使缺锌FTase(apo-FTase)完全失活，但不能使全酶失活。用碘乙酰胺处理该酶后，也发现了类似的效果。在apo-FTase中加入锌可以保护它不被NEM失活，这些发现表明存在特定的半胱氨酸残基(S)，可能位于锌结合部位，是FTase活性所必需的。我们实施了选择性标记策略，用[H-3]NEM修饰从酶中去除锌后暴露的半胱氨酸残基。将修饰后的酶用胰酶消化，对4个标记肽进行鉴定和测序，其中1个为主要标记部位，其余3个标记程度较低。主标记肽含有放射性标记的半胱氨酸残基Cys(299)，它位于FTase的β亚基中，在所有已知的蛋白质戊基转移酶中都是保守的。通过定点突变将半胱氨酸残基转变为丙氨酸和丝氨酸，并在大肠杆菌中表达和纯化突变蛋白，两种突变蛋白都保留了与法尼基二磷酸结合的能力，但基本上失去了所有的催化活性和结合锌的能力。这些结果表明，FTaseβ亚基中的半胱氨酸(299)在酶的催化作用中起着关键作用，可能是该酶中直接配位锌原子的残基之一。
Protein farnesyltransferase (FTase) is a zinc metalloenzyme that performs a post-translational modification on many proteins that is critical for their function. The importance of cysteine residues in FTase activity was investigated using cysteine-specific reagents. Zinc-depleted FTase (apo-FTase), but not the holoenzyme, was completely inactivated by treatment with N-ethylmaleimide (NEM). Similar effects were detected after treatment of the enzyme with iodoacetamide. The addition of zinc to apo-FTase protects it from inactivation by NEM, These findings indicated the presence of specific cysteine residue(s), potentially located at the zinc binding site, that are required for FTase activity. We performed a selective labeling strategy whereby the cysteine residues exposed upon removal of zinc from the enzyme were modified with [H-3]NEM. The enzyme so modified was digested with trypsin, and four labeled peptides were identified and sequenced, one peptide being the major site of labeling and the remaining three labeled to lesser extents. The major labeled peptide contained a radiolabeled cysteine residue, Cys(299), that is in the beta subunit of FTase and is conserved in all known protein prenyltransferases. This cysteine residue was changed to both alanine and serine by site-directed mutagenesis, and the mutant proteins were produced in Escherichia coli and purified, While both mutant proteins retained the ability to bind farnesyl diphosphate, they were found to have lost essentially all catalytic activity and ability to bind zinc. These results indicate that the Cys(299) in the beta subunit of FTase plays a critical role in catalysis by the enzyme and is likely to be one of the residues that directly coordinate the zinc atom in this enzyme.