CHARACTERIZATION OF THE CATALYTIC RESIDUES OF THE TOBACCO ETCH VIRUS 49-KDA PROTEINASE
CHARACTERIZATION OF THE CATALYTIC RESIDUES OF THE TOBACCO ETCH VIRUS 49-KDA PROTEINASE
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DOI：
10.1016/0042-6822(89)90132-3
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发表时间：
1989-09-01
期刊：
影响因子：
3.7
通讯作者：
FLETTERICK, RJ
中科院分区：
文献类型：
--
作者：
DOUGHERTY, WG;PARKS, TD;FLETTERICK, RJ
烟草蚀纹病毒 (TEV) 的 49 kDa 蛋白酶在五个位置裂解源自 TEV 基因组 RNA 的多蛋白。对 49-kDa TEV 蛋白酶进行分子遗传学和生化分析，以测试其与细胞胰蛋白酶样丝氨酸蛋白酶的同源性。包含 TEV 49-kDa 蛋白酶基因和侧翼序列的 cDNA 片段在无细胞转录/翻译系统中表达，并导致形成经过快速自我加工的多蛋白前体。使用定点诱变来测试改变单个 49-kDa 氨基酸残基对蛋白水解的影响。数据表明，TEV 49-kDa 蛋白酶上的催化三联体可能由 His234、Asp269 和 Cys339 组成。这些发现与以下假设一致：TEV 49-kDa 蛋白酶在结构上与类胰蛋白酶家族的丝氨酸蛋白酶相似，但替换了 Cys339 作为活性位点亲核体。 TEV 49-kDa 蛋白酶的结构模型提出了活性位点三联体和底物结合袋附近的其他病毒特异性差异。该结构可以解释大多数细胞蛋白酶抑制剂对这种病毒蛋白酶活性的影响可以忽略不计。
The 49-kDa proteinase of tobacco etch virus (TEV) cleaves the polyprotein derived from the TEV genomic RNA at five locations. Molecular genetic and biochemical analyses fo the 49-kDa TEV proteinase were performed to test its homology to the cellular trypsin-like serine proteases. A cDNA fragment, containing the TEV 49-kDa proteinase gene and flanking sequences, was expressed in a cell-free transcription/translation system and resulted in the formation of a polyprotein precursor that underwent rapid self-processing. Site-directed mutagenesis was used to test the effect of altering individual 49-kDa amino acid residues on proteolysis. The data suggest that the catalytic triad on the TEV 49-kDa proteinase could be composed of the His234, Asp269, and Cys339. These findings are consistent with the hypothesis that the TEV 49-kDa proteinase is structurally similar to the trypsin-like family of serine proteinases with the substitution of Cys339 as the active site nucleophile. A structural model of the TEV 49-kDa proteinase proposes other virus-specific differences in the vicinity of the active site triad and substrate-binding pocket. The structure may explain the observed negligible effect of most cellular proteinase inhibitors on the activity of this viral proteinase.