Intersubunit location of the active site of mammalian ornithine decarboxylase as determined by hybridization of site-directed mutants.
The active form of mammalian ornithine decarboxylase (ODC) is a homodimer consisting of two monomer subunits of 53 kDa each. We have used in vitro hybridization of two different catalytically inactive mutants of ODC to determine whether in the wild-type enzyme each monomer contains an independent active site or whether the active sites are shared at the interfaces between the two subunits. Two distinct mutants were obtained using oligonucleotide-directed mutagenesis: In one, cysteine-360, the major alpha-(difluoromethyl)ornithine (alpha-DFMO, a suicide inhibitor of ODC) binding site was converted to alanine. In the other, lysine-69, the pyridoxal 5'-phosphate (PLP, the cofactor of ODC) binding residue was converted to alanine. Expression of each mutant, in vitro, in reticulocyte lysate translation mix, results in the production of a completely inactive enzyme. In contrast, their coexpression restores enzymatic activity to about 25% of the wild-type enzyme. Moreover, coexpression of wild-type subunits with monomers containing both inactivating mutations reduced their activity to about 25%, while their coexpression with monomers that contain a single inactivating mutation reduced the activity to 50%. Cross-linking analysis has demonstrated that activity restoration and repression are both fully correlated with the formation of heterodimers between mutant subunits and between mutant and wild-type subunits, respectively. We therefore conclude that the active site of ODC is formed at the interface of the two monomers through the interaction of the cysteine-360-containing region of one monomer subunit with the region that contains lysine-69 of the other subunit.
Department of Molecular Genetics and Virology, Weizmann Institute of Science, Rehovot, Israel.
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Tobias
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Kahana
D000409:Alanine; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine; D000518:Eflornithine / Q000378:metabolism; D008239:Lysine; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009693:Nucleic Acid Hybridization; D009955:Ornithine Decarboxylase / Q000737:chemistry* / Q000235:genetics; D011732:Pyridoxal Phosphate / Q000378:metabolism; D013329:Structure-Activity Relationship
D046911:Macromolecular Substances; D011732:Pyridoxal Phosphate; D009955:Ornithine Decarboxylase; D008239:Lysine; D003545:Cysteine; D000409:Alanine; D000518:Eflornithine