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)

Glutamic acid 553 of Pseudomonas aeruginosa exotoxin A (ETA) was identified earlier as a putative active-site residue by photoaffinity labeling with NAD. Here ETA-E553D, a cloned form of the toxin in which Glu-553 has been replaced by aspartic acid, was purified from Escherichia coli extracts and characterized. Cytotoxicity of the mutant toxin for mouse L-M cells was less than 1/400,000 that of the wild type. The mutation caused a 3200-fold reduction in NAD:elongation factor 2 ADP-ribosyltransferase activity, as estimated by assays with an active fragment derived from the toxin by digestion with thermolysin. NAD glycohydrolase activity was reduced somewhat less, by a factor of 50, and photoaffinity labeling with NAD by a factor of 2. We detected less than 2-fold change in the values of KM for NAD or elongation factor 2 and no change in KD for NAD, as determined by quenching of protein fluorescence. The drastic reduction of ADP-ribosyltransferase activity therefore results primarily from an effect of the mutation on kcat, implying that Glu-553 plays an important and possibly direct role in catalyzing this reaction. The effects of the E553D mutation are similar to those of the E148D mutation in diphtheria toxin, supporting the notion that these two Glu residues perform the same function in their respective toxins.
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PMID:Pseudomonas aeruginosa exotoxin A: alterations of biological and biochemical properties resulting from mutation of glutamic acid 553 to aspartic acid. 197 45

Pseudomonas toxin is produced as a proenzyme which is cytotoxic for cells in culture but must be activated to express full enzymatic activity. The ability of purified pseudomonas alkaline protease and elastase or of culture filtrates of two strains of Pseudomonas aeruginosa to modify the activity of pseudomonas toxin was examined. Two parameters of toxin activity were followed: enzymatic activity, i.e., the adenosine diphosphate (ADP) ribosylation of elongation factor 2, and biological activity, i.e., inhibition of protein synthesis in cultured mouse fibroblasts. Biological activity of toxin depends upon an intact toxin molecule, whereas enzyme activity requires only a functional A region. Incubation with purified pseudomonas proteolytic enzymes did not alter either enzymatic or biological activity. The toxin is not refractory to the action of all proteolytic enzymes, since thermolysin rapidly destroyed the toxin molecule. Treatment of toxin with culture filtrates of P. aeruginosa reduced ADP ribosylation activity, but increased the ability of toxin to inhibit protein synthesis in cell monolayers. Incubation of culture filtrates with one of the protease inhibitors alpha-2-macroglobulin or phosphoramidon did not alter the effect of the filtrates on biological activity. Alpha-2-macroglobulin, however, caused a fourfold stimulation of ADP ribosylation activity of the toxin. We conclude that pseudomonas alkaline protease and elastase are not responsible for the modifications in toxin activity induced by culture filtrates of P. aeruginosa; the factors responsible have not yet been identified, but are not inactivated by phosphoramidon or alpha-2-macroglobulin.
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PMID:Resistance of exotoxin A to purified Pseudomonas proteolytic enzymes. 615 7