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Protein Engineering, Vol. 14, No. 12, 993-1000, December 2001
© 2001 Oxford University Press

Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency

Rachel B. Kapust, József Tözsér1, Jeffrey D. Fox, D.Eric Anderson,2, Scott Cherry, Terry D. Copeland and David S. Waugh,3

Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, PO Box B, Frederick, MD 21702-1201, USA and 1 Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Hungary

Because of its stringent sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (TEV) is a useful reagent for cleaving genetically engineered fusion proteins. However, a serious drawback of TEV protease is that it readily cleaves itself at a specific site to generate a truncated enzyme with greatly diminished activity. The rate of autoinactivation is proportional to the concentration of TEV protease, implying a bimolecular reaction mechanism. Yet, a catalytically active protease was unable to convert a catalytically inactive protease into the truncated form. Adding increasing concentrations of the catalytically inactive protease to a fixed amount of the wild-type enzyme accelerated its rate of autoinactivation. Taken together, these results suggest that autoinactivation of TEV protease may be an intramolecular reaction that is facilitated by an allosteric interaction between protease molecules. In an effort to create a more stable protease, we made amino acid substitutions in the P2 and P1' positions of the internal cleavage site and assessed their impact on the enzyme's stability and catalytic activity. One of the P1' mutants, S219V, was not only far more stable than the wild-type protease (~100-fold), but also a more efficient catalyst.


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