DOMAIN ORGANIZATION AND A PROTEASE-SENSITIVE LOOP IN EUKARYOTIC ORNITHINE DECARBOXYLASE

Trypanosoma brucei ornithine decarboxylase was reconstituted by coexpr ession of two polypeptides corresponding to residues 1-305 and residue s 306-425 in Escherichia coli. The two peptides were coexpressed, at w ild-type levels, from a single transcriptional unit that was separated by a 15-nucleotide untranslated region containing a ribosome binding site. The fragmented enzyme was purified and analyzed. The N- and C-te rminal peptides are tightly associated into a fully active tetramer wh ich has the same molecular weight as the native dimer. The kinetic con stants (K-m and k(cat)) measured for the decarboxylation of ornithine are identical to those obtained for the wild-type enzyme. These result s suggest that the enzyme is organized into two structural domains, wi th a domain boundary in the region of amino acid 305. In contrast, the individual N- and C-terminal peptides are expressed primarily as incl usion bodies. Small quantities of soluble N-terminal peptide could be purified. This truncated protein is capable of inhibiting the wild-typ e enzyme, suggesting that it is folded into a native-like structure. L imited proteolysis with trypsin or chymotrypsin identifies a likely su rface loop at amino acids 160-170, present in both the mouse and T. br ucei enzyme, which positions one or more functionally important active site residues (e.g., Lys169). Kinetic analysis of a chimeric enzyme c omposed of T. brucei and mouse ornithine decarboxylase suggests that t he substrate carboxylate binding determinant is located between residu es 1 and 170.