Dissecting a charged network at the active site of orotidine-5 '-phosphatedecarboxylase

The crystal structure of yeast orotidine-5'-phosphate decarboxylase in comp lex with the postulated transition state analog, 6-hydroxyuridine-5'-phosph ate, reveals contacts between this inhibitor and a novel quartet of charged residues (Lys-59, Asp-91, Lys-93, and Asp-96) within the active site. The structure also suggests a possible interaction between O-2 of the 6-hydroxy uridine-5'-phosphate pyrimidine ring and Gln-215, Here we report the result s of mutagenesis of each of the charged active site residues and Gln-215, T he activities of the Q215A and wild-type enzymes were equal indicating that any interactions between this residue and the pyrimidine ring are dispensa ble for efficient decarboxylation. For the D91A and K93A mutant enzymes, ac tivity was reduced by more than 5 orders of magnitude and substrate binding could not be detected by isothermal calorimetry. For the D96A mutant enzym e, k(cat) was reduced by more than 5 orders of magnitude, and isothermal ca lorimetry indicated an Ii-fold decrease in the affinity of this enzyme for the substrate in the ground state. For the K59A enzyme, k(cat) was reduced by a factor of 130, and K-m had increased by a factor of 900. These results indicate that the integrity of the network of charged residues is essentia l for transition state stabilization.