Biosynthesis, purification, and characterization of the human coronavirus 229E 3C-like proteinase.
Coronavirus gene expression involves proteolytic processing of the gene 1-encoded polyprotein(s), and a key enzyme in this process is the viral 3C-like proteinase. In this report, we describe the biosynthesis of the human coronavirus 229E 3C-like proteinase in Escherichia coli and the enzymatic properties, inhibitor profile, and substrate specificity of the purified protein. Furthermore, we have introduced single amino acid substitutions and carboxyl-terminal deletions into the recombinant protein and determined the ability of these mutant 3C-like proteinases to catalyze the cleavage of a peptide substrate. Using this approach, we have identified the residues Cys-3109 and His-3006 as being indispensable for catalytic activity. Our results also support the involvement of His-3127 in substrate recognition, and they confirm the requirement of the carboxyl-terminal extension found in coronavirus 3C-like proteinases for enzymatic activity. These data provide experimental evidence for the relationship of coronavirus 3C-like proteinases to other viral chymotrypsin-like enzymes, but they also show that the coronavirus proteinase has additional, unique properties.
Ziebuhr
J
J
Institute of Virology, University of Würzburg, Germany. j.ziebuhr@rzbox.uni-wuerzburg.de
Heusipp
G
G
Siddell
S G
SG
D000595:Amino Acid Sequence; D002918:Chymotrypsin / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D017934:Coronavirus / Q000201:enzymology*; D028941:Coronavirus 229E, Human; D010450:Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D012697:Serine Endopeptidases; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D011994:Recombinant Proteins; D010450:Endopeptidases; D012697:Serine Endopeptidases; D002918:Chymotrypsin