Ke. Tobias et C. Kahana, INTERSUBUNIT LOCATION OF THE ACTIVE-SITE OF MAMMALIAN ORNITHINE DECARBOXYLASE AS DETERMINED BY HYBRIDIZATION OF SITE-DIRECTED MUTANTS, Biochemistry, 32(22), 1993, pp. 5842-5847
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.