CHARACTERIZATION OF NLA PROTEASE FROM TURNIP MOSAIC POTYVIRUS EXHIBITING A LOW-TEMPERATURE OPTIMUM CATALYTIC ACTIVITY

Nuclear inclusion protein a (NIa) protease of turnip mosaic potyvirus is responsible for the processing of the viral polyprotein into functi onal proteins. The NIa protease was found to exhibit its optimum catal ytic activity at approximately 15 degrees and a bell-shaped pH-depende nt activity profile with a maximum at approximately pH 8.5. Kinetic st udies showed that both Km and K-max values were lower at 12 than at 25 degrees in all three different pH conditions, pH 7.0, 7.4, and 8.3, i ndicating that the higher activity al 12 degrees is due to the lower v alue of K-m. Interestingly, the self-cleavage of the 27-kDa protease t o generate the 25-kDa protease occurred more rapidly al 25 than at 12 degrees, implying that the C-terminal self-cleavage site may interfere with the binding of the peptide substrate to the active site of the p rotease. Mutations and deletions at the C-terminal cleavage site had n o effect on the temperature dependence of the proteolytic activity, de monstrating that the C-terminal self-cleavage is not related to the lo w-temperature optimum catalytic activity. The fluorescence measurement of the NIa protease upon temperature variation revealed that the prot ease undergoes a large conformational change between 2 and 42 degrees and a drastic transition near 45 degrees, suggesting that the low-temp erature optimum catalytic activity is due to the highly flexible struc ture of the NIa protease. (C) 1996 Academic Press, Inc.