Mammalian Pitrilysin: Substrate Specificity and Mitochondrial Targeting

Abstract

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 Km values of 2 μM or less. Three of four peptides containing 11 or fewer residues exhibited Km values above 10 μM. Similarly, peptides containing 13 or more residues exhibited kcat values below 10 min −1 , while three of four peptides containing 11 or fewer residues exhibited kcat 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 does however 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 GFP, this 15-amino acid N-terminal sequence directed the fusion protein to the mitochondria.Zinc-dependent peptidases of the M16 family possess the active site motif HEXXH in which the two histidine residues coordinate a zinc atom and the glutamate facilitates attack of a water molecule on the scissile bond. A subgroup of the M16 family of metallopeptidases contains an inverted Zn-binding motif HXXEH and these peptidases are referred to as inverzincins (1). The first identified inverzincin was pitrilysin protease III (2), a gene product of the ptr gene in E. coli.Besides protease III (EC 3.4.24.55), several mammalian inverzincins have been identified and classified as M16A family members (3). These include insulysin (insulin degrading enzyme, IDE, EC 3.4.24.56) and nardilysin (NRDc, EC 3.4.24.61). Both protease III and insulysin exhibit a broad substrate specificity cleaving a variety of peptides. The structures of pitrilysin protease III and insulysin (4) are very similar and appear to be "clam-shaped" with the insulysin structure in a closed conformation and the protease III structure in an open conformation. The closed conformation tightly locks down the substrate in the interior of the enzyme, requiring a conformational change to permit product release. This conformational change is likely the Address correspondence to: Louis B. Hersh, Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, B283 BBSRB, 741 South Limestone St., Lexington, Fax: (859) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript ratelimiting step for the insulysin reaction. Both of these enzymes have an active site pocket that can accommodate and interact with a variety of peptides, explaining in part their broad substrate specificity. In contrast, nardilysin has a more defined substrate specificity cleaving peptides primarily at or between dibasic p...

Alzheimer's disease is associated with altered expression of genes involved in immune response and mitochondrial processes in astrocytes

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Targeting Capacity and Conservation of PreP Homologues Localization in Mitochondria of Different Species

“…1. 3,10,16,22 To elucidate the targeting capacity of the presequences, we fused the predicted presequence, together with the N-terminal portion of the mature proteins (AtPreP and hPreP, the first 40 amino acids; Cym1, the first 33 amino acids), to GFP. The fusion constructs were translated in a coupled transcription/translation reaction in the presence of radiolabeled methionine and imported into mitochondria isolated from spinach, rat, and yeast ( Fig.…”

Short Forms of Ste20-related Proline/Alanine-rich Kinase (SPAK) in the Kidney Are Created by Aspartyl Aminopeptidase (Dnpep)-mediated Proteolytic Cleavage

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