Chemical rescue of I-site cleavage in living cells and in vitro discriminates between the cytomegalovirus protease, assemblin, and its precursor, pUL80a.
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 approximately 11%/h in bacteria and approximately 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.
McCartney
Stephen A
SA
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Brignole
Edward J
EJ
Kolegraff
Keli N
KN
Loveland
Amy N
AN
Ussin
LaShon M
LM
Gibson
Wade
W
D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D002460:Cell Line; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D005240:Feasibility Studies; D005998:Glycine / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D020539:Sequence Analysis, Protein; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D007093:Imidazoles; C029899:imidazole; D012697:Serine Endopeptidases; C071910:assemblin; D000409:Alanine; D005998:Glycine