Chemical rescue of I-site cleavage in living cells and in vitro discriminates between the cytomegalovirus protease, assemblin, and its precursor, pUL80a.
Chemical rescue of I-site cleavage in living cells and in vitro discriminates between the cytomegalovirus protease, assemblin, and its precursor, pUL80a.
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活细胞和体外 I 位点裂解的化学拯救可区分巨细胞病毒蛋白酶、组装蛋白及其前体 pUL80a。
DOI：
10.1074/jbc.m506876200
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Gibson,Wade
中科院分区：
文献类型：
--
作者：
McCartney,StephenA;Brignole,EdwardJ;Kolegraff,KeliN;Loveland,AmyN;Ussin,LaShonM;Gibson,Wade
化学拯救是一种既定的方法，它为设计突变酶提供了一种定向策略，其中可以通过提供适当的外源化合物来恢复活性。这种方法已成功地用于体外研究广泛的酶，但其应用于生命系统受到的关注较少。我们已经研究了使用化学拯救来制造巨细胞病毒（CMV）成熟蛋白酶的条件致死突变体的可行性。28-kDa CMV丝氨酸蛋白酶assemblin具有Ser-His-His催化三联体和靠近其中点的内部（I）切割位点。我们发现，咪唑可以恢复I-位点切割失活的突变体取代关键活性位点的丙氨酸或甘氨酸，这挽救更好。在人和猴CMV装配蛋白同源物的对应突变体中观察到类似的拯救，并且在活细胞和体外都发生。通过氨基酸测序确定切割在正确的位点，并且在细菌中以约11%/h进行，在体外以约30%/h进行。在组装蛋白前体pUL 80 a的情况下，相同的突变对化学拯救无反应。这种催化差异区分了CMV蛋白酶的两种形式。
Chemical rescue is an established approach that offers a directed strategy for designing mutant enzymes in which activity can be restored by supplying an appropriate exogenous compound. This method has been used successfully to study a broad range of enzymesin vitro, but its application to living systems has received less attention. We have investigated the feasibility of using chemical rescue to make a conditional-lethal mutant of the cytomegalovirus (CMV) maturational protease. The 28-kDa CMV serine protease, assemblin, has a Ser-His-His catalytic triad and an internal (I) cleavage site near its midpoint. We found that imidazole can restore I-site cleavage to mutants inactivated by replacing the critical active site His with Ala or with Gly, which rescued better. Comparable rescue was observed for counterpart mutants of the human and simian CMV assemblin homologs and occurred in both living cells andin vitro. Cleavage was established to be at the correct site by amino acid sequencing and proceeded at ∼11%/h in bacteria and ∼30%/hin vitro. The same mutations were unresponsive to chemical rescue in the context of the assemblin precursor, pUL80a. This catalytic difference distinguishes the two forms of the CMV protease.