Acidic residues important for substrate binding and cofactor reactivity in eukaryotic ornithine decarboxylase identified by alanine scanning mutagenesis.
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.
Osterman
A L
AL
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235, USA.
Kinch
L N
LN
Grishin
N V
NV
Phillips
M A
MA
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
C093486:N-methylpyridoxal-5'-phosphate; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D011732:Pyridoxal Phosphate; D001120:Arginine; D009952:Ornithine; D009955:Ornithine Decarboxylase; D008239:Lysine