Chemical rescue of I-site cleavage in living cells and in vitro discriminates between the cytomegalovirus protease, assemblin, and its precursor, pUL80a

Stephen A. McCartney, Edward J. Brignole, Keli N. Kolegraff, Amy N. Loveland, LaShon M. Ussin, D Wade Gibson

Research output: Contribution to journalArticle

Abstract

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 enzymes in 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 and in vitro. Cleavage was established to be at the correct site by amino acid sequencing and proceeded at ∼11%/h in bacteria and ∼30%/h in 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.

Original languageEnglish (US)
Pages (from-to)33206-33212
Number of pages7
JournalJournal of Biological Chemistry
Volume280
Issue number39
DOIs
StatePublished - Sep 30 2005

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Cytomegalovirus
Peptide Hydrolases
Cells
Serine Proteases
Enzymes
Protein Sequence Analysis
Bacteria
Amino Acids
Catalytic Domain
Mutation
assemblin
In Vitro Techniques

ASJC Scopus subject areas

  • Biochemistry

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Chemical rescue of I-site cleavage in living cells and in vitro discriminates between the cytomegalovirus protease, assemblin, and its precursor, pUL80a. / McCartney, Stephen A.; Brignole, Edward J.; Kolegraff, Keli N.; Loveland, Amy N.; Ussin, LaShon M.; Gibson, D Wade.

In: Journal of Biological Chemistry, Vol. 280, No. 39, 30.09.2005, p. 33206-33212.

Research output: Contribution to journalArticle

McCartney, Stephen A. ; Brignole, Edward J. ; Kolegraff, Keli N. ; Loveland, Amy N. ; Ussin, LaShon M. ; Gibson, D Wade. / Chemical rescue of I-site cleavage in living cells and in vitro discriminates between the cytomegalovirus protease, assemblin, and its precursor, pUL80a. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 39. pp. 33206-33212.
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