Abstract
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 (approximately 100-fold), but also a more efficient
catalyst.
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