Characterization of active-site residues of the NIa protease from tobacco vein mottling virus

Nuclear inclusion a (NIa) protease of tobacco vein mottling virus is respon sible for the processing of the viral polyprotein into functional proteins. In order to identify the active-site residues of the TVMV NIa protease, th e putative active-site residues, His-46, Asp-81 and Cys-151, mere mutated i ndividually to generate H46R, H46A, D81E, D81N, C151S, and C151A, and their mutational effects on the proteolytic activities were examined, Proteolyti c activity,vas completely abolished by the mutations of H45R, H46A, D81N, a nd C151A, suggesting that the three residues are crucial for catalysis. The mutation of D81E decreased k(cat) marginally by about 4.7-fold and increas ed K-m by about 8-fold, suggesting that the aspartic acid at position 81 is important for substrate binding hut can be substituted by glutamate withou t any significant decrease in catalysis, The replacement of Cys-151 by Ser to mimic the catalytic triad of chymotrypsinlike serine protease resulted i n the drastic decrease in k(cat) by about 1,260-fold. This result might be due to the difference of the active-site geometry between the NIa protease and chymotrypsin. The protease exhibited a bell-shaped pH-dependent profile with a maximum activity approximately at pH 8.3 and with the abrupt change s 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 res ults demonstrate that the three residues, His-46, Asp-81, and Cys-151, play a crucial role in catalysis of the TVMV NIa protease.