The K12 side chain runs across the surface of TIM, and the side chain cation interacts with substrate bound beneath the protein surface. The E97 side chain lies in the second shell of active site side chains and forms an anchoring ion pair to the first shell K12 side chain cation (Figure ). This side chain is conserved at the active sites of all TIMs. We have carried out EVB simulations to model the effects of amino acid substitutions of E97, in order to examine the functional role that mandates conservation of anchoring the K12•E97 ion pair at TIM. .