Mutational analysis of the putative catalytic triad of the cowpea mosaic virus 24K protease.
To investigate the mechanism of action of the cowpea mosaic virus (CPMV) 24K protease, a full-length cDNA clone of bottom component (B) RNA has been constructed from which RNA can be transcribed in vitro using T7 RNA polymerase. Translation of the resulting RNA in rabbit reticulocyte lysate leads to the synthesis of a 200 kDa product (the 200K protein) which cleaves itself in a manner identical to that of the product translated from B RNA isolated from virions. Site-directed mutagenesis of the full-length clone was used to examine the effects of altering individual amino acids in the 24K protease on its activity. The results obtained are consistent with the prediction that the 24K protease is structurally similar to the trypsin-like family of serine proteases and suggest that His40, Glu76, and Cys166 comprise the active site. Substitution of Cys166 by a serine residue results in an enzyme with reduced catalytic activity.
Dessens
J T
JT
Department of Virus Research, John Innes Institute, Norwich, United Kingdom.
Lomonossoff
G P
GP
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D007887:Fabaceae; D005814:Genes, Viral; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000201:enzymology / Q000235:genetics*; D009841:Oligonucleotides / Q000737:chemistry; D010946:Plants, Medicinal; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics
D009841:Oligonucleotides; D011506:Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; D004247:DNA; D010450:Endopeptidases; C070745:24K protease, cowpea mosaic virus