LOCATIONS AND FUNCTIONAL ROLES OF CONSERVED LYSINE RESIDUES IN SALMONELLA-TYPHIMURIUM OROTATE PHOSPHORIBOSYLTRANSFERASE

Salmonella typhimurium orotate phosphoribosyltransferase (OPRTase) catalyzes the formation of orotidine 5'-monophosphate (OMP) from orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). There are five highly conserved lysine residues (Lys-19, -26, -73, -100, and -103) in S. typhimurium OPRTase. Here, we report the results of mutagenesis and substrate analog studies to investigate the functional roles of these lysines. Together with information from X-ray crystallography [Scapin, G., Grubmeyer, C., and Sacchettini, J. C. (1994) Biochemistry 33, 1287-1294; Scapin, G., Ozturk, D. H., Grubmeyer, C., and Sacchettini, J. C. (1995) Biochemistry 34, 10744-10754], sequence comparisons, and chemical modification [Grubmeyer, C., Segura, E., and Dorfman, R. (1993) J. Biol. Chem. 268, 20299-20304], this work permits the assignment of functions of the five conserved lysines. Lys-19 is external to the active site, and its mutation to glutamine had little effect on enzyme activity. Lys-26 forms a hydrogen bond to OMP at the 3'-hydroxyl group, and its mutation produced 3-10-fold decreases in k(cat). Lys-73 extends into the active site, and a conformational change allows it to interact with either the 5'-phosphate of OMP or the 2-hydroxyl and alpha-phosphoryl oxygen of PRPP in their respective substrate complexes. Mutation of Lys-73 produced a 50-100-fold decrease in k(cat) and an 8-12-fold increase in the K-M value for PRPP. Mutation of Lys-100 produced a 5-fold decrease in k(cat) and a 3-fold increase in the K-M for PRPP, consistent with its location within the active site, near the pyrophosphate moiety of PRPP. Lys-103, which is conserved among all known OPRTases, does not show any interaction with PRPP or OMP in enzyme ligand complexes, and K-M values for these ligands exhibited relatively minor perturbations in Lys-103 mutants. However, mutation of Lys-103 produced 600-1000-fold decreases in k(cat), implying an essential role in catalysis.