Active site mutations in CheA, the signal-transducing protein kinase of the chemotaxis system in XXXX .

@entity1025 @entity70639 @entity526 @entity70633 @entity3793 @entity70636 @entity616 @entity70644 @entity70634 @entity70641 @entity70638 @entity70643 @entity70640 @entity70642 @entity70635 @entity70637 @entity855
We investigated the functional roles of putative active site residues in @entity526 CheA by generating nine site-directed mutants, purifying the mutant proteins, and quantifying the effects of those mutations on autokinase activity and binding affinity for @entity855 . We designed these mutations to alter key positions in sequence motifs conserved in the protein @entity3793 kinase family, including the N box (H376 and N380), the G1 box (D420 and G422), the F box (F455 and F459), the G2 box (G470, G472, and G474), and the " @entity616 " (T499), a motif identified by comparison of CheA to members of the GHL family of ATPases. Four of the mutant CheA proteins exhibited no detectable autokinase activity (Kin(-)). Of these, three ( @entity70633 , @entity70634 , and @entity70635 ) exhibited moderate decreases in their affinities for @entity855 in the presence or absence of @entity1025 . The other Kin(-) mutant ( @entity70636 / @entity70637 / @entity70638 ) exhibited wild-type affinity for @entity855 in the absence of @entity1025 , but reduced affinity (relative to that of wild-type CheA) in the presence of @entity1025 . The other five mutants (Kin(+)) autophosphorylated at rates slower than that exhibited by wild-type CheA. Of these, three mutants ( @entity70639 , @entity70640 , and @entity70641 / @entity70642 ) exhibited severely reduced k(cat) values, but preserved K(M)( @entity855 ) and K(d)( @entity855 ) values close to those of wild-type CheA. Two mutants ( @entity70643 and @entity70644 ) exhibited only small effects on k(cat) and K(M)( @entity855 ). Overall, these results suggest that conserved residues in the N box, G1 box, G2 box, and F box contribute to the @entity855 binding site and autokinase active site in CheA, while the @entity616 makes little, if any, contribution. We discuss the effects of specific mutations in relation to the three-dimensional structure of CheA and to binding interactions that contribute to the stability of the complex between CheA and @entity1025 -bound @entity855 in both the ground state and the transition state for the CheA autophosphorylation reaction.
