Acidic residues important for substrate binding and cofactor reactivity in eukaryotic ornithine decarboxylase identified by alanine scanning mutagenesis

J Biol Chem. 1995 May 19;270(20):11797-802. doi: 10.1074/jbc.270.20.11797.


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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Arginine / chemistry*
  • Base Sequence
  • Binding Sites
  • Eukaryotic Cells / enzymology*
  • Kinetics
  • Leishmania donovani / enzymology
  • Lysine / chemistry*
  • Mice / metabolism
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed*
  • Ornithine / chemistry*
  • Ornithine Decarboxylase / chemistry*
  • Ornithine Decarboxylase / genetics
  • Protozoan Proteins / chemistry*
  • Protozoan Proteins / genetics
  • Pyridoxal Phosphate / analogs & derivatives
  • Pyridoxal Phosphate / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • Species Specificity
  • Substrate Specificity
  • Trypanosoma brucei brucei / enzymology
  • Trypanosoma brucei brucei / genetics


  • N-methylpyridoxal-5'-phosphate
  • Protozoan Proteins
  • Recombinant Fusion Proteins
  • Pyridoxal Phosphate
  • Arginine
  • Ornithine
  • Ornithine Decarboxylase
  • Lysine