Effects of engineering complementary charged residues into the hydrophobic subunit interface of tyrosyl-tRNA synthetase

Abstract

Wild-type tyrosyl-tRNA synthetase (TyrTS) from Bacillus stearothermophilus is a symmetrical dimer. Four different heterodimeric enzymes have been produced by site-directed mutagenesis at the subunit interface so that the monomers are linked by a potential salt bridge in a hydrophobic environment. The two Phe-164 residues of wild-type TyrTS are on the axis of symmetry and interact in a hydrophobic region of the subunit interface. Mutation of Phe-164 to aspartate or glutamate in full-length TyrTS and to lysine or arginine in an active truncated enzyme (.DELTA.TyrTS) induces reversible dissociation of the enzyme into inactive monomers. Mixing mutants in equimolar amounts produces four different heterodimers: TyrTS(Asp-164)-.DELTA.TyrTS(Lys-164); TyrTS(Asp-164)-.DELTA.TyrTS(Arg-164); TyrTS(Glu-164)-.DELTA.TyrTS(Lys-164); TyrTS(Glu-164)-.DELTA.TyrTS(Arg-164). A general method is derived for analyzing the kinetics of dimeric enzymes that reversibly dissociate into inactive subunits. Application to mutants of TyrTS allows estimation of dissociation constants (Kd values) for the dimers. At pH 7.8, the heterodimers have Kd values of 6-14 .mu.M, whereas for homodimers Kd = 120-4000 .mu.M. These values decrease to about 30 .mu.M for homodimers of TyrTS(Asp-164), TyrTS(Glu-164), and .DELTA.TyrTS(Lys-164) when the pH favors uncharged forms of the side chains at position 164. Each of the four salt bridges engineered into the hydrophobic subunit interface of TyrTS appears, therefore, to be weak. These engineered salt bridges may be compared with naturally occurring ones. In the latter, there are complementary interactions between the charges in the salt bridge with polar groups in the protein. The potential salt bridges engineered into TyrTS were not designed to be stabilized in this way. They are consequently weak but are sufficient to direct specificity in dimerization.