Nuclear inclusion a (NIa) protease of tobacco vein mottling virus is responsible for the processing of the viral polyprotein into functional proteins. In order to identify the active-site residues of the TVMV NIa protease, the putative active-site residues, His-46, Asp-81 and Cys-151, were mutated individually to generate H46R, H46A, D81E, D81N, C151S, and C151A, and their mutational eects on the proteolytic activities were examined. Proteolytic activity was completely abolished by the

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... of H46R, H46A, D81N, and C151A, suggesting that the three residues are crucial for catalysis. The mutation of D81E decreased k cat marginally by about 4.7-fold and increased K m by about 8-fold, suggesting that the aspartic acid at position 81 is important for substrate binding but can be substituted by glutamate without any signi®cant decrease in catalysis. The replacement of Cys-151 by Ser to mimic the catalytic triad of chymotrypsinlike serine protease resulted in the drastic decrease in k cat by about 1,260-fold. This result might be due to the dierence of the active-site geometry between the NIa protease and chymotrypsin. The protease exhibited a bell-shaped pH-dependent pro®le with a maximum activity approximately at pH 8.3 and with the abrupt changes at the respective pK a values of approximately 6.6 and 9.2, implying the involvement of a histidine residue in catalysis. Taken together, these results demonstrate that the three residues, His-46, Asp-81, and Cys-151, play a crucial role in catalysis of the TVMV NIa protease.