Study on substrate specificity at subsites for severe acute respiratory syndrome coronavirus 3CL protease.
Autocleavage assay and peptide-based cleavage assay were used to study the substrate specificity of 3CL protease from the severe acute respiratory syndrome coronavirus. It was found that the recognition between the enzyme and its substrates involved many positions in the substrate, at least including residues from P4 to P2'. The deletion of either P4 or P2' residue in the substrate would decrease its cleavage efficiency dramatically. In contrast to the previous suggestion that only small residues in substrate could be accommodated to the S1' subsite, we have found that bulky residues such as Tyr and Trp were also acceptable. In addition, based on both peptide-based assay and autocleavage assay, Ile at the P1' position could not be hydrolyzed, but the mutant L27A could hydrolyze the Ile peptide fragment. It suggested that there was a stereo hindrance between the S1' subsite and the side chain of Ile in the substrate. All 20 amino acids except Pro could be the residue at the P2' position in the substrate, but the cleavage efficiencies were clearly different. The specificity information of the enzyme is helpful for potent anti-virus inhibitor design and useful for other coronavirus studies.
Shan
Yu-Fei
YF
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Xu
Gen-Jun
GJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D000086782:Coronavirus 3C Proteases; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004792:Enzyme Precursors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004792:Enzyme Precursors; D010455:Peptides; D014764:Viral Proteins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases