Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
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