Temperature and salt effects on proteolytic function of turnip mosaic potyvirus nuclear inclusion protein a exhibiting a low-temperature optimum activity

The nuclear inclusion protein a (NIa) of turnip mosaic potyvirus is a prote ase responsible for processing the viral polyprotein into functional protei ns. The NIa protease exhibits an unusual optimum proteolytic activity at ab out 16 degrees C. In order to understand the origin of the low-temperature optimum activity, the effects of temperature and salt ions on the catalytic activity and the structure of the NIa protease have been investigated. The analysis of the temperature dependence of k(cat) and K-m revealed that K-m decreases more drastically than k(cat) as temperature decreases. The therm odynamic analysis showed that the decrease of K-m is driven entropically, s uggesting a possibility that the substrate binding might need a large entro py cost. The secondary structure of the NIa protease was significantly pert urbed at temperatures between 20 and 40 degrees C and the protease was unfo lded at very low concentrations of guanidine hydrochloride with a transitio n midpoint of 0.8 M. These results suggest that the NIa protease is highly flexible in structure. Interestingly, salt ions including NaCl, KCl, CaCl2 and MgCl2 stimulated the proteolytic activity by 2-6-fold and increased the optimum temperature to 20-25 degrees C. This stimulatory effect of the sal t ions was due to the lowering of K-m. The salt ions promoted the structura l rigidity as evidenced in the higher resistance to the heat-induced unfold ing in the presence of the salt ions. The increase in rigidity may lead to the lowering of K-m possibly by reducing the entropic cost for substrate bi nding. Taken together, these results suggest that the NIa protease is highl y flexible in structure and the low-temperature optimum activity might poss ibly be attributed to lowered entropy cost for substrate binding at lower t emperatures. (C) 2000 Elsevier Science B.V. All rights reserved.