The Glu54 residue of the norovirus 3C-like protease was implicated in proteolysis as a third-member carboxylate of the catalytic triad. The E54L mutant protease cleaved the sequence (133)LSFE/AP between the 3B and 3C regions of norovirus polyprotein, but did not cleave the sequence (198)ATSE/GK between the 3A and 3B. The 3BC junction mutation (3B-L133A or 3B-F135S) hampered the cleavage by the E54L protease, whereas the 3AB junction mutation (3A-A198L, S200F) allowed the E54L protease to digest. These results indicate that the E54L mutant protease is a substrate-specificity mutant and requires large hydrophobic amino acid residues at both P4 and P2 positions of the substrate. It was notable that the 3A-S200F P2 position mutation caused tight interaction between the wild-type protease and the C-terminus of the 3A protein, hence a decreased release rate of the product from the enzyme. This tight binding was dependent on the hydrophobicity of amino acid residues introduced at position 200 of the 3A region and was affected by the mutation in the bII-cII loop of the protease or the mutation of position 198 of 3A corresponding to the P4 position of the substrate. These results suggest that the protease and the substrate sense each other in the process of the proteolysis, being supported by crystal structures.