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., & Sacchettini, J. C. (1994) Biochemistry 33, 1287-1294; Scapin, G., Ozturk, D. H., Grubmeyer, C., & Sacchettini, J. C. (1995) Biochemistry 34, 10744-10754], sequence comparisons, and chemical modification [Grubmeyer, C., Segura, E., & 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 kcat. 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 kcat and an 8-12-fold increase in the KM value for PRPP. Mutation of Lys-100 produced a 5-fold decrease in kcat and a 3-fold increase in the KM for PRPP, consistent with its location within the active site, near the pyrophosphate moiety of PRPP.(ABSTRACT TRUNCATED AT 250 WORDS)
Ozturk
D H
DH
Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Dorfman
R H
RH
Scapin
G
G
Sacchettini
J C
JC
Grubmeyer
C
C
D001483:Base Sequence; D001665:Binding Sites; D003196:Computer Graphics; D011756:Diphosphates / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D004795:Enzyme Stability; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009962:Orotate Phosphoribosyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012486:Salmonella typhimurium / Q000201:enzymology; D013379:Substrate Specificity; D014542:Uridine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism
D011756:Diphosphates; D004791:Enzyme Inhibitors; C005417:orotidylic acid; D010754:Phosphoribosyl Pyrophosphate; D014542:Uridine Monophosphate; D009962:Orotate Phosphoribosyltransferase; D008239:Lysine