Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.55 (PTR)
 433 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A periplasmic insulin-cleaving proteinase (ICP), purified to its electrophoretic homogeneity in the SDS-PAGE from the Gram-negative bacterium Acinetobacter calcoaceticus, was examined and compared in its properties with the protease III (protease Pi, pitrilysin, EC 3.4.99.44) of Escherichia coli and the insulin-destroying proteinase (IDE, insulinase, EC 3.4.99.45) from eucaryotes. The enzyme was proven to be a metalloprotease like protease III and IDE, as was shown by the inhibitory effects exerted by EDTA and o-phenanthroline. Furthermore, dialysis against EDTA and o-phenanthroline led to a complete loss of activity, which could be restored by addition of Co2+, and, to a lesser extent, but at a lower metal ion concentration by Zn2+. Similar to protease III and IDE, ICP prefers the cleavage of small polypeptides (insulin, insulin B-chain, glucagon) to the cleavage of proteins (casein, human serum albumin, globin) and was inactive against synthetic amino acid derivates (esters, p-nitranilides, and furoylacroleyl substrates) of subtilisin, thermolysin, trypsin, and chymotrypsin. The peptide-bond-specificity of the ICP in the cleavage of the oxidized insulin B-chain was investigated and the results were compared to the specificity of protease III of E. coli, IDE, protease-24,11, and thermolysin. Cleavage sites in the oxidized insulin B-chain generated by ICP are Asn3-Gln4, His10-Leu11, Ala14-Leu15, Leu17-Val18, Gly23-Phe24, Phe24-Phe25, and Phe25-Tyr26. Principally, ICP cleaves between hydrophobic amino acids and amides. The ICP shares one of the only two cleavage sites with the protease III and four sites with the IDE.
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PMID:A periplasmic insulin-cleaving proteinase (ICP) from Acinetobacter calcoaceticus sharing properties with protease III from Escherichia coli and IDE from eucaryotes. 773 84

Insulin-degrading enzyme (IDE), a nonlysosomal metalloprotease involved in metabolizing internalized insulin, has catalytic properties that have been strongly conserved through evolution. Two major properties distinguish IDE from the prototypic metalloprotease thermolysin. 1) It is inhibited by cysteine protease inhibitors as well as metalloprotease inhibitors; 2) it contains an inversion of the HEXXH active site motif of thermolysin, where the histidines coordinate zinc and the glutamate participates in catalysis. Furthermore, cysteine is adjacent to the glutamate residue (HXCEH) in human, rat, and Drosophila IDE, although it is not conserved in their close homologue, Escherichia coli protease III. This cysteine has been postulated to mediate the differential sensitivity of IDE and protease III to cysteine protease inhibitors and chelators. The role of the cysteine in IDE catalysis and inhibitor sensitivity was examined by mutating Cys110 to glycine or serine. To determine whether glutamate in this unusual motif participates in catalysis, we mutated Glu111 to aspartate, valine, or glutamine. Vectors containing wild type or mutant enzymes were transfected into COS cells, and expression was confirmed by Western blotting. Although the glutamate mutants were devoid of insulin degrading activity, the cysteine mutants were indistinguishable from wild type enzyme in both catalytic activity and sensitivity to inhibitors. The loss of activity in the glutamate mutants was not due to gross alterations in tertiary structure, as shown by retention of the ability to bind substrate and by conservative and nonconservative mutation of a neighboring residue with no apparent effect on catalysis. These results demonstrate that the conserved glutamate in the zinc-binding site of human insulin-degrading enzyme is a major catalytic residue, while a conserved cysteine in this region is not essential for catalysis or inhibitor sensitivity.
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PMID:Functional analysis of conserved residues in the active site of insulin-degrading enzyme. 810 41

We have recently isolated and identified a novel mitochondrial metalloprotease, pre-sequence protease (PreP) from potato and shown that it degrades mitochondrial pre-sequences. PreP belongs to the pitrilysin protease family and contains an inverted zinc-binding motif. To further investigate the degradation of targeting peptides, we have overexpressed the Arabidopsis thaliana homologue of PreP, zinc metalloprotease (Zn-MP), in Escherichia coli. We have characterized the recombinant Zn-MP with respect to its catalytic site, substrate specificity and intracellular localization. Mutagenesis studies of the residues involved in metal binding identified the histidines and the proximal glutamate as essential residues for the proteolytic activity. Substrate specificity studies showed that the Zn-MP has the ability to degrade both mitochondrial pre-sequences and chloroplastic transit peptides, as well as other unstructured peptides. The Zn-MP does not recognize an amino acid sequence per se. Immunological studies and proteolytic activity measurements in isolated mitochondria and chloroplasts revealed the presence of the Zn-MP in both organelles. Furthermore, the Zn-MP was found to be dually imported to both mitochondria and chloroplasts in vitro. In summary, our data show that the Zn-MP is present and serves the same function in chloroplasts as in mitochondria--degradation of targeting peptides.
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PMID:Characterization of a novel zinc metalloprotease involved in degrading targeting peptides in mitochondria and chloroplasts. 1461 63

Pitrilysin is a bacterial protease that is similar to the mammalian insulin-degrading enzyme, which is hypothesized to protect against the onset of Alzheimer's disease, and the yeast enzymes Axl1p and Ste23p, which are responsible for production of the a-factor mating pheromone in Saccharomyces cerevisiae. The lack of a phenotype associated with pitrilysin deficiency has hindered studies of this enzyme. Herein, we report that pitrilysin can be heterologously expressed in yeast such that it functionally substitutes for the shared roles of Axl1p and Ste23p in pheromone production, resulting in a readily observable phenotype. We have exploited this phenotype to conduct structure-function analyses of pitrilysin and report that residues within four sequence motifs that are highly conserved among M16A enzymes are essential for its activity. These motifs include the extended metalloprotease motif, a second motif that has been hypothesized to be important for the function of M16A enzymes, and two others not previously recognized as being important for pitrilysin function. We have also established that the two self-folding domains of pitrilysin are both required for its proteolytic activity. However, pitrilysin does not possess all the enzymatic properties of the yeast enzymes since it cannot substitute for the role of Axl1p in the repression of haploid invasive growth. These observations further support the utility of the yeast system for structure-function and comparative studies of M16A enzymes.
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PMID:A common genetic system for functional studies of pitrilysin and related M16A proteases. 1672 21

The novel peptidasome, called presequence protease, PreP, was originally identified and characterized in Arabidopsis thaliana as a mitochondrial matrix and chloroplast stroma localized metalloprotease. PreP has a function as the organellar peptide clearing protease and is responsible for degrading free targeting peptides and also other unstructured peptides up to 65 amino acid residues that might be toxic to organellar functions. PreP contains an inverted Zn-binding motif and belongs to the pitrilysin protease family. The crystal structure of AtPreP refined at 2.1 A demonstrated a unique totally enclosed large cavity of 10000 A3 that opens and closes in response to peptide binding, revealing a novel catalytic mechanism for proteolysis. Homologues of PreP have been found in yeast and human mitochondria. Interestingly, the human PreP, hPreP, is the protease that is responsible for clearing the human brain mitochondria from the toxic amyloid-beta peptide (Abeta) associated with Alzheimer's disease (AD). Accumulation of Abeta has been shown in the brain mitochondria from AD patients and mutant transgenic mice overexpressing Abeta. Here, we present a review of our present knowledge on structural and functional characteristics of PreP and discuss its mitochondrial Abeta-degrading activity in the human brain mitochondria in relation to AD.
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PMID:The organellar peptidasome, PreP: a journey from Arabidopsis to Alzheimer's disease. 2003 33