Glu(191) and Asp(195) in rat mitochondrial processing peptidase beta subunit are involved in effective cleavage of precursor protein through interaction with the proximal arginine.
Mitochondrial processing peptidase (MPP), consisting of alpha and beta subunits, recognizes a large variety of N-terminal extension peptides of mitochondrial precursor proteins, and generally cleaves a single site of the peptide including arginine at the -2 position (P(2)). We obtained evidence that Glu(191) and Asp(195) of rat beta subunit interact with P(2) arginine of precursor protein through ionic and hydrogen bonds, respectively, using recombinant MPP. Mutation to alanines at Glu(191) and Asp(195) reduced processing activity toward precursors with P(2) arginine, but resulted in no loss of activity toward P(2) alanine precursors. Charge-complementary mutation demonstrated that MPP variants with beta Arg(191) exhibited compensatory processing activity for the precursor with acidic residue at the P(2) position. Thus, Glu(191) and Asp(195) are substrate-binding sites required for cleavage of extension peptides through interaction with P(2) arginine.
Kitada
S
S
Department of Chemistry, Kyushu University, Fukuoka, 812-8581, Japan. s.kitscc@mbox.nc.kyushu-u.ac.jp
Kojima
K
K
Ito
A
A
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001120:Arginine / Q000737:chemistry*; D001224:Aspartic Acid / Q000737:chemistry*; D001665:Binding Sites; D020134:Catalytic Domain; D004591:Electrophoresis, Polyacrylamide Gel; D005973:Glutamine / Q000737:chemistry*; D008666:Metalloendopeptidases / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D000096122:Mitochondrial Processing Peptidase
D000409:Alanine; D000596:Amino Acids; D001120:Arginine; D001224:Aspartic Acid; D005973:Glutamine; D008666:Metalloendopeptidases; D010455:Peptides; D011994:Recombinant Proteins; D000096122:Mitochondrial Processing Peptidase