A strategically positioned cation is crucial for efficient catalysis by chorismate mutase

Combinatorial mutagenesis and in vivo selection experiments previously affo rded functional variants of the AroH class Bacillus subtilis chorismate mut ase lacking the otherwise highly conserved active site residue Arg(90). Her e, we present a detailed kinetic and crystallographic study of several such variants. Removing the arginine side chain (R90G and R90A) reduced catalyt ic efficiency by more than 5 orders of magnitude. Reintroducing a positive charge to the active site through lysine substitutions restored more than a factor of a thousand in k(cat) Remarkably, the lysine could be placed at p osition 90 or at the more remote position 88 provided a sterically suitable residue was present at the partner site. Crystal structures of the double mutants C88S/R90K and C88K/R90S show that the lysine adopts an extended con formation that would place its E-ammonium group within hydrogen-bonding dis tance of the ether oxygen of bound chorismate in the transition state. Thes e results provide support for the hypothesis that developing negative charg e in the highly polarized transition state is stabilized electrostatically by a strategically placed cation. The implications of this finding for the mechanism of all natural chorismate mutases and for the design of artificia l catalysts are discussed.