Identification of residues in ornithine decarboxylase essential for enzymic activity and for rapid protein turnover.
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.
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Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033.
Stanley
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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
D009841:Oligonucleotides; D009955:Ornithine Decarboxylase