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 homodi mer consisting of two monomer subunits of 53 kDa each. We have used in vitro hybridization of two different catalytically inactive mutants o f ODC to determine whether in the wild-type enzyme each monomer contai ns an independent active site or whether the active sites are shared a t the interfaces between the two subunits. Two distinct mutants were o btained using oligonucleotide-directed mutagenesis: In one, cysteine-3 60, the major alpha-(difluoromethyl)ornithine (alpha-DFMO, a suicide i nhibitor of ODC) binding site was converted to alanine. In the other, lysine-69, the pyridoxal 5'-phosphate (PLP, the cofactor of ODC) bindi ng residue was converted toalanine. Expression of each mutant, in vitr o, in reticulocyte lysate translation mix, results in the production o f a completely inactive enzyme. In contrast, their coexpression restor es enzymatic activity to about 25% of the wild-type enzyme. Moreover, coexpression of wild-type subunits with monomers containing both inact ivating mutations reduced their activity to about 25%, while their coe xpression with monomers that contain a single inactivating mutation re duced the activity to 50%. Cross-linking analysis has demonstrated tha t activity restoration and repression are both fully correlated with t he formation of heterodimers between mutant subunits and between mutan t and wild-type subunits, respectively. We therefore conclude that the active site of ODC is formed at the interface of the two monomers thr ough the interaction of the cysteine-360-containing region of one mono mer subunit with the region that contains lysine-69 of the other subun it.