MPP, a dinner of non-identical subunits, is the primary peptidase responsible for the removal of leader peptides from nuclearly coded mitochondrial proteins. Alignments of the α and β subunits of MPP from different species show high homologies in certain regions, including a highly negatively charged region and a region containing the putative metal ion binding site - HFLEK in the o-subunit and HFLEH in the β-subunit. We generated variant enzyme subunits by site-directed rnutagenesis, expressed them in Ecoli, and determined their proteolytic activity in vitro. In the putative metal ion binding site in the a-subunit, when we substitute K, F, or R for H109, the activity decreased to less than 5%. Interestingly, the activity was only moderately reduced (to -30%) by the substitution K113H. When we substituted R for H101 or H105 or inverted the binding motif to HEXXH in the β-subunit, the activity decreased to less than 1%. We detected slightly higher (about 3%) activity in the substitution H101Q. Substitution E104D had very little effect on MPP activity, while E104Q yielded completely inactive enzyme. In the negatively charged region of the a-subunit, when we changed E179 to Q, we detected less than 3% activity. In the β-subunit, by changing acidic amino acids to basic ones and vice versa between Q162 and D188, we narrowed the crucial amino acid residues at the site of the predicted a-helix to a stretch of eight (E174 through E181). Substitution of D or Q for E174 or E181 produced enzyme with decreased activity.
|Original language||English (US)|
|State||Published - 1996|
ASJC Scopus subject areas
- Molecular Biology