Mammalian pitrilysin: substrate specificity and mitochondrial targeting.
The substrate specificity of the mitochondrial metallopeptidase proteinase 1 (MP1) was investigated and its mitochondrial targeting signal identified. The substrate specificity of MP1 was examined with physiological peptides as substrates. Although the enzyme exhibits broad substrate specificity, there is a trend for peptides containing 13 or more residues to exhibit K(m) values of 2 muM or less. Three of four peptides containing 11 or fewer residues exhibited K(m) values above 10 muM. Similarly, peptides containing 13 or more residues exhibited k(cat) values below 10 min(-1), while three of four peptides containing 11 or fewer residues exhibited k(cat) values above 30 min(-1). Many of the peptide cleavage sites of MP1 resemble that of the mitochondrial processing protease (MPP); however, MP1 does not process the precursor form of citrate synthase. The enzyme, however, does cleave the released prepeptide from precitrate synthase. A mitochondria localization was shown in MP1 transfected NT2 and HepG2 cells. Deletion of the N-terminal 15 amino acids caused MP1 to be mislocalized to the cytoplasm and nucleus. Furthermore, when fused to green flourescent protein, this 15-amino acid N-terminal sequence directed the fusion protein to the mitochondria.
Chow
K Martin
KM
Department of Molecular and Cellular Biochemistry, University of Kentucky, B283 BBSRB, 741 South Limestone Street, Lexington, Kentucky 40536-0509, USA.
Gakh
O
O
Payne
I C
IC
Juliano
Maria Aparecida
MA
Juliano
Luiz
L
Isaya
G
G
Hersh
Louis B
LB
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002950:Citrate (si)-Synthase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D007477:Ions; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology*; D008969:Molecular Sequence Data; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology; D013379:Substrate Specificity / Q000187:drug effects; D014443:Tyrosine / Q000378:metabolism; D001615:beta-Endorphin / Q000378:metabolism
D007477:Ions; D008670:Metals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D001615:beta-Endorphin; D002950:Citrate (si)-Synthase; D008666:Metalloendopeptidases; C046583:pitrilysin