| Abstract
| - The crystal structures of orotidine 5‘-monophosphate decarboxylases from four different sourceshave been published recently. However, the detailed mechanism of catalysis of the most proficient enzymeknown to date remains elusive. As the ligand−protein interactions at the orotate binding site are crucialto the understanding of this enzyme, we mutated several of the residues surrounding the aromatic part ofthe substrate, individually and in combination. The ensuing effects on enzyme structure and stabilitywere characterized by X-ray crystallography of inhibitor, product, or substrate complexes and by chemicaldenaturation with guanidine hydrochloride, respectively. The results are consistent with the residuesK42D70K72D75B being charged and forming an ‘alternate charge network' around the reactive part of thesubstrate. In addition to exerting charge−charge repulsion on the orotate carboxylate, Asp70 also makesa crucial contribution to enzyme stability. Consequently, orotidine 5‘-monophosphate decarboxylases seemto require the presence of a negative charge at this position for catalysis as well as for correct and stablefolding.
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