A model for enzyme-substrate interaction in alanine racemase

We report on a theoretical model for the complex of the enzyme alanine race mase with its natural substrate (L-alanine) and cofactor (pyridoxal 5'-phos phate). Electrostatic potentials were calculated and ionization states were predicted for all of the ionizable groups in alanine racemase. Some rather unusual charge states were predicted for certain residues. Tyr265 ' has an unusually low predicted pK(a) of 7.9 and at pH 7.0 has a predicted average charge of -0.37, meaning that 37% of the Tyr265 ' residues in an ensemble of enzyme molecules are in the phenolate form. At pH 8-9, the majority of T yr265 ' side groups will be in the phenolate form. This lends support to th e experimental evidence that Tyr265 ' is the catalytic base involved in the conversion of L-alanine to D-alanine. Residues Lys39 and Lys129 have predi cted average charges of +0.91 and +0.14, respectively, at pH 7.0. Lys39 is believed to be the catalytic base for the conversion of D-alanine to L-alan ine, and the present results show that, at least some of the time, it is in the unprotonated amine form and thus able ra act as a base. Cys311 ', whic h is located very close to the active site, has an unusually low predicted pK(a) of 5.8 and at pH 7.0 has a predicted average charge of -0.72. The ver y low predicted charge for Lys129 is consistent with experimental evidence that it is carbamylated, since an unprotonated amine group is available to act as a Lewis base and form the carbamate with CO2. Repeating the pK(a) ca lculations on the enzyme with Lys129 in carbamylated form predicts trends s imilar to those of the uncarbamylated enzyme. It appears that the enzyme ha s the ability to stabilize negative charge in the region of the active site . Implications for selective inhibitor design are discussed.