Chemical Rescue of I-site Cleavage in Living Cells and in Vitro Discriminates between the Cytomegalovirus Protease, Assemblin, and Its Precursor, pUL80a
作者： Stephen A. McCartney , Edward J. Brignole , Keli N. Kolegraff , Amy N. Loveland , LaShon M. Ussin
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摘要: Chemical rescue is an established approach that offers a directed strategy for designing mutant enzymes in which activity can be restored by supplying appropriate exogenous compound. This method has been used successfully to study broad range of vitro, but its application living systems received less attention. We have investigated the feasibility using chemical make conditional-lethal cytomegalovirus (CMV) maturational protease. The 28-kDa CMV serine protease, assemblin, Ser-His-His catalytic triad and internal (I) cleavage site near midpoint. found imidazole restore I-site mutants inactivated replacing critical active His with Ala or Gly, rescued better. Comparable was observed counterpart human simian assemblin homologs occurred both cells vitro. Cleavage at correct amino acid sequencing proceeded ∼11%/h bacteria ∼30%/h same mutations were unresponsive context precursor, pUL80a. difference distinguishes two forms
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