title: Acidic residues important for substrate binding and cofactor reactivity in eukaryotic ornithine decarboxylase identified by alanine scanning mutagenesis.
abstract: Ornithine decarboxylases from Trypanosoma brucei, mouse, and Leishmania donovani share strict specificity for three basic amino acids, ornithine, lysine, and arginine. To identify residues involved in this substrate specificity and/or in the reaction chemistry, six conserved acidic resides (Asp-88, Glu-94, Asp-233, Glu-274, Asp-361, and Asp-364) were mutated to alanine in the T. brucei enzyme. Each mutation causes a substantial loss in enzyme efficiency. Most notably, mutation of Asp-361 increases the Km for ornithine by 2000-fold, with little effect on kcat, suggesting that this residue is an important substrate binding determinant. Mutation of the only strictly conserved acidic residue, Glu-274, decreases kcat 50-fold; however, substitution of N-methylpyridoxal-5'-phosphate for pyridoxal-5'-phosphate as the cofactor in the reaction restores the kcat of E274A to wild-type levels. These data demonstrate that Glu-274 interacts with the protonated pyridine nitrogen of the cofactor to enhance the electron withdrawing capability of the ring, analogous to Asp-222 in aspartate aminotransferase (Onuffer, J. J., and Kirsch, J. F. (1994) Protein Eng. 7, 413-424). Eukaryotic ornithine decarboxylase is a homodimer with two shared active sites. Residues 88, 94, 233, and 274 are contributed to each active site from the same subunit as Lys-69, while residues 361 and 364 are part of the Cys-360 subunit.
authors.affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235, USA.
authors.forename: A L
authors.initials: AL
authors.lastname: Osterman
authors.forename: L N
authors.initials: LN
authors.lastname: Kinch
authors.forename: N V
authors.initials: NV
authors.lastname: Grishin
authors.forename: M A
authors.initials: MA
authors.lastname: Phillips
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D005057:Eukaryotic Cells / Q000201:enzymology*; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology; D008239:Lysine / Q000737:chemistry*; D051379:Mice / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009952:Ornithine / Q000737:chemistry*; D009955:Ornithine Decarboxylase / Q000737:chemistry* / Q000235:genetics; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013045:Species Specificity; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000235:genetics
chemical_list: C093486:N-methylpyridoxal-5'-phosphate; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D011732:Pyridoxal Phosphate; D001120:Arginine; D009952:Ornithine; D009955:Ornithine Decarboxylase; D008239:Lysine