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

Because of its stringent sequence specificity, the catalyticdomain of the nuclear inclusion protease from tobacco etch virus(TEV) is a useful reagent for cleaving genetically engineeredfusion proteins. However, a serious drawback of TEV proteaseis that it readily cleaves itself at a specific site to generatea truncated enzyme with greatly diminished activity. The rateof autoinactivation is proportional to the concentration ofTEV protease, implying a bimolecular reaction mechanism. Yet,a catalytically active protease was unable to convert a catalyticallyinactive protease into the truncated form. Adding increasingconcentrations of the catalytically inactive protease to a fixedamount of the wild-type enzyme accelerated its rate of autoinactivation.Taken together, these results suggest that autoinactivationof TEV protease may be an intramolecular reaction that is facilitatedby an allosteric interaction between protease molecules. Inan effort to create a more stable protease, we made amino acidsubstitutions in the P2 and P1' positions of the internal cleavagesite and assessed their impact on the enzyme's stability andcatalytic activity. One of the P1' mutants, S219V, was not onlyfar more stable than the wild-type protease (~100-fold), butalso a more efficient catalyst.