Glutamate Residues Required for Substrate Binding and Cleavage Activity in Mitochondrial Processing Peptidase

Abstract

Mitochondrial processing peptidase, a metalloendopeptidase consisting of ␣-and ␤-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 ␤-subunit, which has a putative metal-binding motif, H 56 XXEH 60 , 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 ␣-subunit. Most nuclear-encoded mitochondrial proteins are synthesized on cytoplasmic ribosomes as larger precursors with Nterminal extension peptides for targeting into mitochondria (1-3). During or after import of the precursors into mitochondria, the extension peptides are proteolytically removed by three types of processing peptidases. Mitochondrial processing peptidase (MPP 1 ; EC 3.4.24.64) (4 -8) generally cleaves off a large part of the extension peptide, including the mitochondrial matrix-targeting sequence as the initial step of the processing. Many precursors are converted into the mature forms by the one-step processing. The second enzyme is mitochondrial intermediate peptidase, which catalyzes second-step cleavage in the two-step processing of some precursor proteins (9, 10). These precursors are first cleaved by MPP, and then the residual octapeptides are removed by the mitochondrial intermediate peptidase. The last enzyme is inner membrane protease I, which processes the signal sequence for inner membrane and intermembrane space (11). The last two peptidases sequentially act after cleavage of the matrix targeting sequences by MPP. Thus, MPP plays an important role in proteolytical processing of precursors in mitochondria. MPP has been purified from mitochondria of Neurospora crassa (12), yeast Saccharomyces cerevisiae (13), rat liver (14), and a few plants (15,16). The enzymes, except for those of the plant, are soluble proteins in the mitochondrial matrix and consist of ␣ and ␤ subunits. The subunits from yeast and rat liver form a stable heterodimer, whereas Neurospora subunits could be separated from each other by gel filtration. Processing activity of the enzymes is sensitive to metal chelators, and the lost activity is restored by divalent metal ions (4 -7). MPP specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off the extension peptides at single sites (14,17,18). Contrary to the strict substrate specificity of the enzyme, amino acid sequences of the extension peptides are wide in length and poor in similarity (1). Many experimental observations have indicated that basic amino acid residues in the extension peptides were required for the effective processing by . A consensus sequence of the processing signals has...

A Proposed Common Structure of Substrates Bound to Mitochondrial Processing Peptidase

“…The two subunits form a ball with a crack leading to the internal cavity. Functional amino acid residues predicted in our previous works (17)(18)(19) are arranged around the cavity. In the simulation model (Fig.…”

“…In the simulation model (Fig. 6), the distance between Glu-73 in yeast ␤-MPP (corresponding to Glu-104 in rat ␤-MPP), which is a catalytic center (17,18), and Glu-390/Asp-391 in yeast ␣-MPP (corresponds to Glu-446/Asp-447 in rat ␣-MPP), which we assumed to be residues interacting with the distal arginine in the presequence (19), was calculated to be about 30 Å. The glycine-rich loop in ␣-MPP, which is conserved among different organisms and has been shown to be essential for MPP function, is close to the metal binding active center in ␤-MPP and is located about 30 Å from Glu-390/Asp-391 in ␣-MPP.…”

“…Glu-174 may participate in the catalytic reaction (18). Glu-124, which is in a characteristic acidic amino acid cluster conserved in ␤-MPP, may interact with the N-terminal portion of the presequence in the cleavage reaction (18). On the other hand, Glu-390 and Asp-391 in yeast ␣-MPP interact with the distal arginine residues, which are required especially for cleavage of precursors with a longer presequence (19).…”

“…Glu-181 is the third metal-binding residue (19). Glu-174 may participate in the catalytic reaction (18). Glu-124, which is in a characteristic acidic amino acid cluster conserved in ␤-MPP, may interact with the N-terminal portion of the presequence in the cleavage reaction (18).…”

“…Some functional amino acid residues in MPP were determined using mutational analysis; His-101, Glu-104, and His-105 in rat ␤-MPP, which form a metal binding site, HxxEH, conserved among a pitrilysin metallopeptidase superfamily (16), are the catalytic center of MPP (17,18). Glu-181 is the third metal-binding residue (19).…”