Characterization of Nla protease from turnip mosaic potyvirus exhibiting a low-temperature optimum catalytic activity

Abstract

The nuclear inclusion protein a {Nla) of turnip mosaic potyvirus is a protease responsible for the processing of the viral polyprotein into functional proteins. The Nia protease had been found to exhibit its optimum catalytic activity at approximately 15 "C. In order to investigate the relationship between the catalytic activity and temperature, kinetic, mutational, and structural studies were carried out. Both Km and V'ma\ values were lower at 12 "C than al 25 "C in all three different pH conditions. pH 7.0. 7.4, and 8.3, indicating that the higher activity at 12 "C is due to the lower value of Km. Interestingly, the self-cleavage of the 27-kDa protease to generate the 25-kDa protease occurred more rapidly at 25 'C than at 12 LrC, implying that the C-terminal self-cleavage site may interfere with the binding of the peptide substrate to the active site of the protease. Mutations at the cleavage site had no effect on the temperature dependence of the proteolytic activity, indicating that the C-terminal self-cleavage is not related to the low-temperature optimum catalytic activity. The fluorescence measurement of the Nia protease upon temperature variation revealed that the protease undergoes a large conformational change between 2 "C and 42 'C and a drastic transition near 45 "C. Unfolding of the Nla protease by guanidine hydrochloride exhibited a transition with a midpoint at 0.8 M of guanidine hydrochloride. These results suggest that the low-temperature optimum catalytic activity is due to the highly flexible structure of the Nla protease.