Abstract
Mitochondrial processing peptidase, a metalloendopeptidase consisting of alpha- and beta-subunits, specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off N-terminal extension peptides. The enzyme requires the basic amino acid residues in the extension peptides for effective and specific cleavage. To elucidate the mechanism involved in the molecular recognition of substrate by the enzyme, several glutamates around the active site of the rat beta-subunit, which has a putative metal-binding motif, H56XXEH60, were mutated to alanines or aspartates, and effects on kinetic parameters, metal binding, and substrate binding of the enzyme were analyzed. None of mutant proteins analyzed was impaired in dimer formation with the alpha-subunit. Mutation of glutamates at positions 79, 129, and 136, in addition to an active-site glutamate at position 59, resulted in a marked decrease in cleavage efficiency. Together with sequence alignment data, glutamate 136 appears to be involved in metal binding. Glutamate 129 is mostly responsible for the catalysis, as there was a considerable decrease in kcat value by the mutation. Mutation of glutamate 79 led to decrease in kcat value and increase in Km values. Substrate binding experiments using an environmentally sensitive fluorescence probe attached to the peptide showed that the mutation caused a remarkable environmental change at the binding site to the N-terminal region of the substrate peptide and decreased binding of the peptide, thereby suggesting that glutamate 79 participates primarily in substrate binding. Thus, some glutamate residues required for substrate binding and cleavage activity have been identified.
Highlights
Most nuclear-encoded mitochondrial proteins are synthesized on cytoplasmic ribosomes as larger precursors with Nterminal extension peptides for targeting into mitochondria [1,2,3]
We have identified amino acid residues involved in the enzyme function, and we attempted to elucidate mechanisms involved in molecular recognition of substrates by the enzyme
Site-directed Mutation of Glutamate Residues in the N-terminal Portion of -MPP—To identify residues responsible for substrate recognition and catalytic reaction, we focused on glutamic acids in the N-terminal region of -MPP, because this region contains a putative active-site sequence, HXXEH, and has a significant degree of homology to members of the pitrilysin family (Fig. 1)
Summary
Most nuclear-encoded mitochondrial proteins are synthesized on cytoplasmic ribosomes as larger precursors with Nterminal extension peptides for targeting into mitochondria [1,2,3]. The second enzyme is mitochondrial intermediate peptidase, which catalyzes second-step cleavage in the two-step processing of some precursor proteins [9, 10]. The subunits of MPP have significant sequence homology with a family of endopeptidases, the pitrilysin family [29, 30], that includes Escherichia coli pitrilysin ( called protease III), the insulin-degrading enzymes from mammals and insects, the N-arginine dibasic convertase from rat. Despite metalloendopeptidases, they lack the thermolysin-like zinc binding motif HEXXH, but all the members except the ␣-MPPs have the inverted motif, HXXEH. We find that some glutamate residues are essential for substrate binding and catalytic reaction
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