OMP decarboxylase : active site labeling using a combination of site-directed mutagenesis and chemical modification

Abstract

Orotidine 5'-monophosphate decarboxylase (ODCase) catalyzes the conversion of OMP to UMP. The enzyme functions without metals or cofactors. It has been proposed that an active site lysine donates a proton to the substrate OMP during decarboxylation. For a lysine to donate a proton, it must have an unusually low pKa. To investigate the Pka of the active site lysines in ODCase from yeast and E. coli, we attempted to specifically label the side chains of these lysines. The K93C and "C93 only" mutants of the yeast enzyme were generated and purified, but resulted in poorly soluble proteins after purification. The mutations C41S C161S C168S and K73C of E. coli ODCase were generated by site directed mutagenesis. The triple mutant, with no cysteine residues present, was insoluble regardless of efforts made to obtain soluble protein. Attempts to modify K73C with DTNB, in order to reversibly protect non-active site cysteines, were unsuccessful. Chemical modifications of K73C with promoethylamine restored partial decarboxylation activity to the inactive mutant protein. The results provide a basis for future efforts to generate mutated, chemically modified ODCase for NMR analysis.