Mapping the Active Site-Ligand Interactions of Orotidine 5'-Monophosphate Decarboxylasae by Crystallography

摘要

The crystal structure of orotidine 5'-monophosphate decarboxylases from four different sources have been published recently. However, the detailed mechanism of catalysis of the most proficient enzyme known to date remains elusive. As the ligand-protein interactions at the orotate binding site are crucial to the understanding of this enzyme, we mutated several of the residues surrounding the aromatic part of the substrate, individually and in combination. The ensuing effects on enzyme structure and stability were characterized by X-ray crystallography of inhibitor, product, or substrate complexes and by chemical denaturation with guanidine hydrochloride, respectively. The results are consistent with the residues K~42D~70K~72D~75B being charged and forming an 'alternative charge network' around the reactive part of the substrate. In addition to exerting charge-charge repulsion on the orotate carboxylate, Asp~70 also makes a crucial contribution to enzyme stability. Consequently, orotidine 5'-monophosphate decarboxylases seem to require the presenceof a negative charge at this position for catalysis as well as for correct and stable folding.