TRUNCATION AND HEME POCKET MUTATIONS REDUCE PRODUCTION OF FUNCTIONAL CATALASE HPII IN ESCHERICHIA-COLI

The subunit of catalase HPII from Escherichia coli is 753 residues in length and contains a core of similar to 500 residues, with high struc tural similarity to all other heme catalases, To this core are added e xtensions of similar to 80 and 180 residues at the N- and C-termini, r espectively. The tetrameric structure is made up of a pair of interwov en dimers in which 90 N-terminal residues of each subunit are inserted through a loop formed by the hinge region linking the beta-barrel and alpha-helical domains of the adjacent subunit, A high concentration o f proline residues is found in the vicinity of the overlap regions, To study the influence of the extended regions on folding and subunit as sociation of HPII, a diversity of modifications have been introduced. Removal of the complete C-terminal domain or the N-terminal extension, either separately or together, effectively creating a small subunit c atalase, resulted in no enzyme accumulation. Systematic truncations sh owed that only nine C-terminal residues (Ile745 to Ala753) could be re moved without significantly affecting the accumulation of active enzym e. Removal or even conservative replacements of the side chain of Arg7 44 significantly reduced the accumulation of active enzyme despite thi s residue interacting only with the C-terminal domain. Removal of as f ew as 18 residues from the N-terminus also reduced accumulation of act ive enzyme, Changes to other residues in the protein, including residu es in the heme binding pocket, also reduced the accumulation of active protein without substantially affecting the enzyme specific activity. Implications of these data for the interdependence of subunit folding and subunit-subunit interactions are discussed.