title: Identification of residues in ornithine decarboxylase essential for enzymic activity and for rapid protein turnover.
abstract: The importance of certain amino acid residues in mammalian ornithine decarboxylase activity and degradation was studied by site-specific mutagenesis. Changes were made to the mouse ornithine decarboxylase cDNA in a plasmid containing a T7 RNA polymerase promoter. The plasmid was then used for the synthesis of RNA, which was translated in a reticulocyte lysate system. The activity of the ornithine decarboxylase formed and the stability of the protein to degradation in a reticulocyte lysate system were determined. Changes of lysine-169 or of histidine-197 to alanine completely abolished enzyme activity, indicating that these residues are essential for enzyme activity. The removal of the C-terminal 36 residues, the mutation of lysine-349 to alanine, of lysine-298 to alanine or the double change of serine-303 and glutamic acid-308 to alanine residues still resulted in an active enzyme. The last-mentioned finding indicates that the phosphorylation of serine-303 does not play an essential role in the catalytic activity of ornithine decarboxylase. The control ornithine decarboxylase protein was degraded rapidly in a reticulocyte lysate provided that ATP was added. The truncated protein missing the 36 residues from the C-terminus was much more stable in this system, and the protein containing the double change of serine-303 and glutamic acid-308 to alanine residues was slightly more stable than control ornithine decarboxylase protein. These results indicate that the altered residues may play a role in interaction with factors responsible for the rapid turnover of ornithine decarboxylase.
authors.affiliation: Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033.
authors.forename: L
authors.initials: L
authors.lastname: Lu
authors.forename: B A
authors.initials: BA
authors.lastname: Stanley
authors.forename: A E
authors.initials: AE
authors.lastname: Pegg
mesh_terms: D000818:Animals; D001483:Base Sequence; D004252:DNA Mutational Analysis; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D009955:Ornithine Decarboxylase / Q000737:chemistry / Q000378:metabolism*; D014176:Protein Biosynthesis; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D009841:Oligonucleotides; D009955:Ornithine Decarboxylase