PROTEOLYTIC ACTIVITY OF PLUM POX VIRUS TOBACCO ETCH VIRUS CHIMERIC NIA PROTEASES
PROTEOLYTIC ACTIVITY OF PLUM POX VIRUS TOBACCO ETCH VIRUS CHIMERIC NIA PROTEASES
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DOI：
10.1016/0014-5793(91)80360-f
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发表时间：
1991-04-09
期刊：
影响因子：
3.5
通讯作者：
LAIN, S
中科院分区：
文献类型：
--
作者：
GARCIA, JA;LAIN, S
已经构建了编码嵌合NI（a）型蛋白酶的质粒，所述嵌合NI（a）型蛋白酶由来自马铃薯Y病毒属李痘病毒（PPV）和烟草蚀纹病毒（TEV）的序列制成。 在大肠杆菌细胞中测定它们对每种病毒的大核包涵体蛋白（NI（B））-衣壳蛋白（CP）连接的蛋白水解活性。 蛋白酶的氨基部分似乎既不参与酶催化也不参与底物识别。 尽管PPV和TEV NI（a）型蛋白酶之间具有很大的同源性，但分子羧基半部分片段的交换通常会导致酶活性急剧下降。 非活性嵌合蛋白酶不干扰由第二质粒表达的PPV野生型蛋白酶的切割。 结果表明，NI（a）蛋白酶的识别和催化位点是紧密相连的，虽然与底物正确相互作用相关的残基可能存在于蛋白质的其他部分，底物特异性的主要决定因素应该位于一个区域，大约，从羧基端的位置30和90之间。 该区域包括PPV的360位或TEV的355位的保守His，其被假定与切割位点的位置-1的Gln相互作用。
Plasmids encoding chimeric NI(a)-type proteases made of sequences from the potyviruses plum pox virus (PPV) and tobacco etch virus (TEV) have been constructed. Their proteolytic activity on the large nuclear inclusion protein (NI(b))-capsid protein (CP) junction of each virus was assayed in Escherichia coli cells. The amino half of the protease seemed to be involved neither in the enzymatic catalysis nor in substrate recognition. In spite of the large homology among the PPV and TEV NI(a)-type proteases, the exchange of fragments from the carboxyl halves of the molecules usually caused a drastic decrease in the enzymatic activity. Inactive chimeric proteases did not interfere with cleavage by PPV wild type protease expressed from a second plasmid. The results suggest that the recognition and catalytic sites of the NI(a) proteases are closely interlinked and, although residues relevant for the correct interaction with the substrate could be present in other parts of the protein, a main determinant for substrate specificity should lie in a region situated, approximately, between positions 30 and 90 from the carboxyl end. This region includes the conservedHis at position 360 of PPV or 355 of TEV, which has been postulated to interact with the Gln at position - 1 of the cleavage sites.