Site-directed mutagenesis of the putative active site residues of 3C proteinase of coxsackievirus B3: evidence of a functional relationship with trypsin-like serine proteinases.
Picornavirus 3C proteinases (3Cpro) are cysteine proteinases but recent sequence analyses have shown that they are related to trypsin-like serine proteinases. Two models of 3Cpro structure have been presented. Both models indicate that residues His40 and Cys147 are members of the catalytic triad but the models differ in the designation of the third member of the catalytic triad, which is assigned as either Glu71 or Asp85. To test the importance of these four residues in the catalytic activity of 3Cpro of coxsackievirus B3, a member of the enterovirus subgroup of the picornavirus family, single amino acid substitutions were introduced at each of the four sites. All of these mutations resulted in the reduction or inactivation of autocatalytic cleavage of the 3C precursor protein expressed in Escherichia coli, suggesting that all of these residues are essential for the proteolytic reaction. The substitution of Cys147 with Ala abolished 3Cpro activity while the mutant in which Cys147 was replaced with Ser retained reduced proteolytic activity both in cis and in trans. Our results strongly support the proposal that Cys147 of 3Cpro functions as a nucleophile analogous to Ser195 of trypsin-like serine proteinases.
Miyashita
K
K
Central Research Laboratories, Maruishi Pharmaceutical Co., Ltd., Osaka, Japan.
Kusumi
M
M
Utsumi
R
R
Katayama
S
S
Noda
M
M
Komano
T
T
Satoh
N
N
D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D029822:Enterovirus B, Human / Q000201:enzymology*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry*; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000737:chemistry; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism; D014764:Viral Proteins
D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D012697:Serine Endopeptidases; D014357:Trypsin; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases