title: Catalytic site interactions in yeast OMP synthase.
abstract: The enigmatic kinetics, half-of-the-sites binding, and structural asymmetry of the homodimeric microbial OMP synthases (orotate phosphoribosyltransferase, EC 2.4.2.10) have been proposed to result from an alternating site mechanism in these domain-swapped enzymes [R.W. McClard et al., Biochemistry 45 (2006) 5330-5342]. This behavior was investigated in the yeast enzyme by mutations in the conserved catalytic loop and 5-phosphoribosyl-1-diphosphate (PRPP) binding motif. Although the reaction is mechanistically sequential, the wild-type (WT) enzyme shows parallel lines in double reciprocal initial velocity plots. Replacement of Lys106, the postulated intersubunit communication device, produced intersecting lines in kinetic plots with a 2-fold reduction of kcat. Loop (R105G K109S H111G) and PRPP-binding motif (D131N D132N) mutant proteins, each without detectable enzymatic activity and ablated ability to bind PRPP, complemented to produce a heterodimer with a single fully functional active site showing intersecting initial velocity plots. Equilibrium binding of PRPP and orotidine 5'-monophosphate showed a single class of two binding sites per dimer in WT and K106S enzymes. Evidence here shows that the enzyme does not follow half-of-the-sites cooperativity; that interplay between catalytic sites is not an essential feature of the catalytic mechanism; and that parallel lines in steady-state kinetics probably arise from tight substrate binding.
authors.affiliation: Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark.
authors.forename: Michael Riis
authors.initials: MR
authors.lastname: Hansen
authors.affiliation: Department of Biochemistry, Temple University School of Medicine, 3307 N Broad St., Philadelphia, PA 19140, USA.
authors.forename: Eric W
authors.initials: EW
authors.lastname: Barr
authors.affiliation: Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark.
authors.forename: Kaj Frank
authors.initials: KF
authors.lastname: Jensen
authors.affiliation: Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark.
authors.forename: Martin
authors.initials: M
authors.lastname: Willemoës
authors.affiliation: Department of Biochemistry, Temple University School of Medicine, 3307 N Broad St., Philadelphia, PA 19140, USA. Electronic address: ctg@temple.edu.
authors.forename: Charles
authors.initials: C
authors.lastname: Grubmeyer
authors.affiliation: Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark.
authors.forename: Jakob R
authors.initials: JR
authors.lastname: Winther
mesh_terms: D020134:Catalytic Domain; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009962:Orotate Phosphoribosyltransferase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014542:Uridine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: C005417:orotidylic acid; D010754:Phosphoribosyl Pyrophosphate; D014542:Uridine Monophosphate; D009962:Orotate Phosphoribosyltransferase
keywords: Catalytic site interactions; Heterodimer; Kinetic mechanism; Ligand binding; Orotate phosphoribosyltransferase; Stopped-flow