Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Porcine alveolar macrophages (AM), as we reported in 1981, inactivate slow-reacting substance of anaphylaxis (SRS-A) in a time- and cell-number-dependent fashion. In the present study, metabolism of synthetic leukotriene D4 (LTD4) to leukotriene E4 (LTE4) by AM was demonstrated (mean conversion, 216 pmoles LTD/10(7) AM/60 min). The metabolism was inhibited by cooling or by removal of calcium ions plus addition of EDTA but not by dinitrophenol or puromycin, lipoxygenase inhibitors, the peroxidase inhibitor aminotriazole, or the hydroxyl ion scavenger benzoic acid. Similarly, although the addition of catalase plus superoxide dismutase significantly augmented (169 +/- 25% control) SRS release from dispersed porcine lung cells, the presence of these enzymes did not prevent LTD4 metabolism by AM. Inhibitors of gamma-glutamyl transpeptidase were also without effect but the dipeptidase inhibitors L-cysteine and dithiothreitol significantly reduced the conversion, as did pretreatment of AM with thermolysin (100 micrograms/ml). These data indicate that an AM dipeptidase, which may at least partly be located on the cell surface, converts LTD4 to the less bioactive LTE4.
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PMID:Metabolism of leukotriene D4 by porcine alveolar macrophages. 632 Jun 96

We sought to develop a peptide library in solution and dynamically screen this library for peptides that would bind to macromolecules of interest. Peptide diversity was achieved in an initial stock solution of peptides by using proteases under conditions in which both hydrolysis and synthesis occurred. As an example, a simple reaction containing YGG, FL and thermolysin resulted in the synthesis of YGGFL as well as many other undefined products. When low molecular weight products of a reaction containing VA, AL, and thermolysin were subsequently exposed to dipeptidase, 7 out of 9 potential dipeptides were observed. Incubation of protease with an hydrolysate of albumin and a radiolabeled peptide resulted in the radiolabel participating in reactions other than simple hydrolysis and, after 24 h, the specific activity of radiolabel was shown by high performance liquid chromatography to disperse to a level that would be necessary in the event of maximum theoretical diversity. When a binding macromolecule was exposed to this system, ligand production was amplified relative to reactions run in the absence of binding macromolecule. This protease-based peptide scrambling and binding system was utilized for the discovery of novel peptides that bind to fibrinogen.
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PMID:Nonspecific protease-catalyzed hydrolysis/synthesis of a mixture of peptides: product diversity and ligand amplification by a molecular trap. 914 Feb 1

VanX, one of the five proteins required for the vancomycin-resistant phenotype in clinically pathogenic Enterococci, is a zinc-containing d-Ala-d-Ala dipeptidase. To identify potential zinc ligands and begin defining the active site residues, we have mutated the 2 cysteine, 5 histidine, and 4 of the 28 aspartate and glutamate residues in the 202 residue VanX protein. Of 10 mutations, 3 cause inactivation and greater than 90% loss of zinc in purified enzyme samples, implicating His116, Asp123, and His184 as zinc-coordinating residues. Homology searches using the 10 amino acid sequence SxHxxGxAxD, in which histidine and aspartate residues are putative zinc ligands, identified the metal coordinating ligands in the N-terminal domain of the murine Sonic hedgehog protein, which also exhibits an architecture for metal coordination identical to that observed in thermolysin from Bacillus thermoproteolyticus. Furthermore, this 10 amino acid consensus sequence is found in the Streptomyces albus G zinc-dependent N-acyl-d-Ala-d-Ala carboxypeptidase, an enzyme catalyzing essentially the same d-Ala-d-Ala dipeptide bond cleavage as VanX, suggesting equivalent mechanisms and zinc catalytic site architectures. VanX residue Glu181 is analogous to the Glu143 catalytic base in B. thermoproteolyticus thermolysin, and the E181A VanX mutant has no detectable dipeptidase activity, yet maintains near-stoichiometric zinc content, a result consistent with the participation of the residue as a catalytic base.
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PMID:Mutational analysis of potential zinc-binding residues in the active site of the enterococcal D-Ala-D-Ala dipeptidase VanX. 926 30

Angiotensin (Ang) I-converting enzyme (ACE) is a member of the gluzincin family of zinc metalloproteinases that contains two homologous catalytic domains. Both the N- and C-terminal domains are peptidyl-dipeptidases that catalyze Ang II formation and bradykinin degradation. Multiple sequence alignment was used to predict His(1089) as the catalytic residue in human ACE C-domain that, by analogy with the prototypical gluzincin, thermolysin, stabilizes the scissile carbonyl bond through a hydrogen bond during transition state binding. Site-directed mutagenesis was used to change His(1089) to Ala or Leu. At pH 7.5, with Ang I as substrate, k(cat)/K(m) values for these Ala and Leu mutants were 430 and 4,000-fold lower, respectively, compared with wild-type enzyme and were mainly due to a decrease in catalytic rate (k(cat)) with minor effects on ground state substrate binding (K(m)). A 120,000-fold decrease in the binding of lisinopril, a proposed transition state mimic, was also observed with the His(1089) --> Ala mutation. ACE C-domain-dependent cleavage of AcAFAA showed a pH optimum of 8.2. H1089A has a pH optimum of 5.5 with no pH dependence of its catalytic activity in the range 6.5-10.5, indicating that the His(1089) side chain allows ACE to function as an alkaline peptidyl-dipeptidase. Since transition state mutants of other gluzincins show pH optima shifts toward the alkaline, this effect of His(1089) on the ACE pH optimum and its ability to influence transition state binding of the sulfhydryl inhibitor captopril indicate that the catalytic mechanism of ACE is distinct from that of other gluzincins.
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PMID:Angiotensin I-converting enzyme transition state stabilization by HIS1089: evidence for a catalytic mechanism distinct from other gluzincin metalloproteinases. 1106 54