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  • Title: Active site lysines in orotate phosphoribosyltransferase.
    Author: Grubmeyer C, Segura E, Dorfman R.
    Journal: J Biol Chem; 1993 Sep 25; 268(27):20299-304. PubMed ID: 8376388.
    Abstract:
    Orotate phosphoribosyltransferase (OPRTase; EC 2.4.2.10) catalyzes the formation of the nucleotide orotidine-5'-monophosphate from orotate and 5-phosphoribosyl-1-pyrophosphate (PRPP). The bacterial enzyme, unlike its mammalian homolog, is monofunctional and is a dimer of M(r) 23,000 subunits. The availability of large amounts of highly purified crystalline Salmonella typhimurium OPRTase have enabled us to being structure/function studies on the enzyme. Like other phosphoribosyltransferases, OPRTase binds the highly charged MgPRPP complex, as well as anionic orotate, suggesting an active site containing basic residues. The S. typhimurium sequence (Scapin, G., Sacchettini, J. C., Dessen, A., Bhatia, M., and Grubmeyer, C. (1993) J. Mol. Biol. 230, 1304-1308) contains 12 lysine and 13 arginine residues, of which Lys-26, Arg-99, Lys-100, Lys-103, and Arg-156 are conserved as identities among the sequences of OPRTases from other organisms, with Lys-19 and Arg-161 replaced by the alternative basic residue in one or more sequences. The lysine modifier 2,4,6-trinitrobenzene sulfonate inactivated S. typhimurium OPRTase in a pseudo first-order process, and OMP and PRPP provided good protection against inactivation. Spectral quantitation of trinitrophenyl (TNP) group incorporation showed that inactivation was correlated with incorporation of one TNP group per subunit. Surprisingly, tryptic proteolysis followed by high performance liquid chromatography and amino acid sequence analysis revealed that four peptides, containing three distinct lysines, had been modified. Peptides modified at Lys-26, Lys-100 and Lys-103, as well as a doubly modified peptide containing TNP groups at Lys-100 and Lys-103, were identified. Inactivation kinetics showed that the 3 lysine residues were modified at equal rates. Protection studies demonstrated that Lys-26, and to a lesser extent Lys-100 and Lys-103, were protected against modification by OMP, whereas PRPP protected Lys-26, Lys-100 and Lys-103. Pyrophosphate protected Lys-100 and Lys-103. The results suggest active site locations for the sequence-conserved and TNP-modified lysine residues, with Lys-26 interacting with the ribose-5-phosphate moiety of OMP and PRPP, and Lys-100 and Lys-103 interacting with the pyrophosphate moiety of PRPP.
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