Characterization of Nla protease from turnip mosaic potyvirus exhibiting a low-temperature optimum catalytic activity.
Nuclear inclusion protein a (Nla) protease of turnip mosaic potyvirus is responsible for the processing of the viral polyprotein into functional proteins. The Nla protease was found to exhibit its optimum catalytic activity at approximately 15 degrees and a bell-shaped pH-dependent activity profile with a maximum at approximately pH 8.5. Kinetic studies showed that both Km and V(max) values were lower at 12 than at 25 degrees in all three different pH conditions, pH 7.0, 7.4, and 8.3, indicating that the higher activity at 12 degrees 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 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 protease. Mutations and deletions at the C-terminal cleavage site had no effect on the temperature dependence of the proteolytic activity, demonstrating that the C-terminal self-cleavage is not related to the low-temperature optimum catalytic activity. The fluorescence measurement of the Nla protease upon temperature variation revealed that the protease undergoes a large conformational change between 2 and 42 degrees and a drastic transition near 45 degrees, suggesting that the low-temperature optimum catalytic activity is due to the highly flexible structure of the Nla protease.
Kim
D H
DH
Department of Life Sciences, Pohang University of Science and Technology, Korea.
Hwang
D C
DC
Kang
B H
BH
Lew
J
J
Choi
K Y
KY
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004279:DNA, Viral; D010450:Endopeptidases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D017800:Potyvirus / Q000201:enzymology*; D011487:Protein Conformation; D013696:Temperature; D014764:Viral Proteins / Q000378:metabolism*
D004279:DNA, Viral; D014764:Viral Proteins; D010450:Endopeptidases; C059992:nuclear inclusion protein a, mosaic viruses