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

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Query: EC:2.4.2.30 (PARP)
13,611 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 Here ETA-E553D, a cloned form of the toxin in which Glu-553 has been replaced by aspartic acid, was purified from Escherichia coli extracts and characterized.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

The specificity of HIV-1 (human immunodeficiency virus-1) protease has been evaluated relative to its ability to cleave the three-domain Pseudomonas exotoxin (PE66) and related proteins in which the first domain has been deleted or replaced by a segment of CD4. Native PE66 is not hydrolyzed by the HIV-1 protease. However, removal of its first domain produces a molecule which is an excellent substrate for the enzyme. The major site of cleavage in this truncated exotoxin, called LysPE40, occurs in a segment that connects its two major domains, the translocation domain (II), and the ADP-ribosyltransferase (III). This interdomain region contains the sequence ...Asn-Tyr-Pro-Thr... which is similar to that surrounding the scissile Tyr-Pro bond in the gag precursor polyprotein, a natural substrate of the HIV-1 protease. Nevertheless, it is not this sequence that is recognized and cleaved by the enzyme, but one 6 residues away, ...Ala-Leu-Leu-Glu... in which the Leu-Leu peptide bond is hydrolyzed. A second, slower cleavage takes place at the Leu-Ala bond 3 residues in from the NH2 terminus of LysPE40. When domain I of PE66 is replaced by a segment comprising the first two domains of CD4, the resulting chimeric protein is hydrolyzed at the same Leu-Leu bond by HIV-1 protease. Enzyme activities toward synthetic peptides modeled after the sequences defined above in LysPE40 are in complete accord, relative to specificity, kinetics, and pH optimum, with results obtained in the hydrolysis of the parent protein. These findings demonstrate that ideas concerning the specificity of the HIV-1 protease that are based solely upon its processing of natural viral polyproteins can be expanded by evaluation of other multidomain proteins as substrates. Moreover, it would appear that it is not a particular conformation, but sequence and accessibility that play the dominant role in defining sites in a protein substrate that are susceptible to hydrolysis by the enzyme.
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PMID:Interdomain hydrolysis of a truncated Pseudomonas exotoxin by the human immunodeficiency virus-1 protease. 210 21

The exotoxin A gene (toxA) from Pseudomonas aeruginosa PAO1 was expressed from the lac promoter in Escherichia coli, and the localization of the toxin A protein was determined. Throughout the growth cycle, the ADP-ribosyltransferase activity of toxin A was gradually reduced in the periplasm of E. coli, with no apparent degradation of the toxin A protein. This suggests the presence of an E. coli periplasmic factor that interferes with the ADP-ribosyltransferase activity in toxin A. Such an inactivating factor was found in the periplasmic extract from control E. coli cells. The processing of toxin A in E. coli was examined by pulse-chase immunoprecipitation experiments. Mature toxin was detected in both the periplasm and cytoplasm, whereas the membranes contained both mature and precursor forms. Toxin A precursor appears to be processed in both the cytoplasm and the periplasm of E. coli. Toxin A proteins from P. aeruginosa PAO1, PA103, and PAK were compared for their secretion in E. coli. Despite the differences in the amino acid sequences of their leader peptides, toxin A proteins from strains PAO1, PA103, and PAK were processed and secreted to the periplasm of E. coli.
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PMID:Secretion of toxin A from Pseudomonas aeruginosa PAO1, PAK, and PA103 by Escherichia coli. 210 26

To be capable of selective killing of tumor cells, the non-selective Pseudomonas aeruginosa exotoxin A must have its cell-binding domain inactivated or removed and then be chemically linked to, or genetically fused with, a specific targeting agent. In the present study, epsilon-NH2 groups of lysine residues of the cell-binding domain of exotoxin A were extensively propionylated with N-succinimidyl-3-propionate (NSP). The NSP-treated exotoxin retained its cytocidal ADP-ribosyltransferase activity, but it could no longer bind to, and inhibit the proliferation of, Friend murine erythroleukemia cells. Cytotoxicity (i.e., the ability to inhibit proliferation) for the Friend erythroid cells was restored completely to the NSP-inactivated exotoxin by conjugating it to ADIF, an autocrine factor secreted by chicken erythroleukemia cells which selectively inhibits the differentiation of erythroid cells such as Friend erythroleukemia cells without inhibiting their proliferation.
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PMID:The cytotoxicity of Pseudomonas exotoxin A, inactivated by modification of the cell-binding domain I, is restored when conjugated to an erythroid cell-specific targeting agent. 210 50

The virally encoded proteases from human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) have been compared relative to their ability to hydrolyze a variant of the three-domain Pseudomonas exotoxin, PE66. This exotoxin derivative, missing domain I and referred to as LysPE40, is made up of a 13-kilodalton NH2-terminal translocation domain II connected by a segment of 40 amino acids to enzyme domain III of the toxin, a 23-kilodalton ADP-ribosyltransferase. HIV protease hydrolyzes two peptide bonds in LysPE40, a Leu-Leu bond in the interdomain region and a Leu-Ala bond in a nonstructured region three residues in from the NH2-terminus. Neither of these sites is cleaved by the AMV enzyme; hydrolysis occurs, instead, at an Asp-Val bond in another part of the interdomain segment and at a Leu-Thr bond in the NH2-terminal region of domain II. Synthetic peptides corresponding to these cleavage sites are hydrolyzed by the individual proteases with the same specificity displayed toward the protein substrate. Peptide substrates for one protease are neither substrates nor competitive inhibitors for the other. A potent inhibitor of HIV type 1 protease was more than 3 orders of magnitude less active toward the AMV enzyme. These results suggest that although the crystallographic models of Rous sarcoma virus protease (an enzyme nearly identical to the AMV enzyme) and HIV type 1 protease show a high degree of similarity, there exist structural differences between these retroviral proteases that are clearly reflected by their kinetic properties.
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PMID:Proteases from human immunodeficiency virus and avian myeloblastosis virus show distinct specificities in hydrolysis of multidomain protein substrates. 216 35

Limited proteolysis of Pseudomonas aeruginosa exotoxin A by four proteases (chymotrypsin, Staphylococcal serine proteinase, pepsin A and subtilisin) resulted in the formation of polypeptides having a molecular mass of approximately 25 kDa. They possessed both enzymatic activity and residual antigenicity. Their N-terminal sequence analysis showed that the different proteases cleaved exotoxin A in a very restricted area within domain Ib (amino acids 365-404). As a result, the polypeptides contained a large portion (13-34 amino acids) of domain Ib linked to the adjacent C-terminal domain III (amino acids 405-613). The major fragment derived from subtilisin cleavage, at a final yield of 35% (S-fragment; residues 392-613; 24201 Da; pI 4.7) possessed the same level of ADP-ribosyltransferase activity as uncleaved exotoxin A (by mass), and a 37-fold higher NAD-glycohydrolase activity. Polyclonal antibodies from rabbits against exotoxin A completely inhibited the ADP-ribosyltransferase activity of both exotoxin A and the S-fragment, but not the NAD-glycohydrolase activity of the S-fragment. Antibodies against the S-fragment neutralized the ADP-ribosyltransferase activity of exotoxin A. These data determine the primary proteolytic cleavage site of exotoxin A, suggest that some residues in the amino acid sequence 392-404 of exotoxin A seem to have a role in binding or positioning elongation factor 2 (EF-2) and show that antibodies recognize the EF-2-binding site but not the NAD(+)-binding site.
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PMID:Biochemical and immunochemical studies of proteolytic fragments of exotoxin A from Pseudomonas aeruginosa. 217 Jan 23

Glutamic acid-553 of Pseudomonas aeruginosa exotoxin A (ETA), identified previously as an active-site residue, was deleted by oligonucleotide-directed mutagenesis of the cloned toxin gene in Escherichia coli. The purified mutant toxin was stable, fully immunoreactive, and capable of blocking toxin receptors. ADP-ribosyltransferase and cytotoxic activities were at least 10(6)-fold lower than those of wild-type ETA, and injection of mice with 50 micrograms (equivalent to 400 lethal doses of ETA) produced no ill effects. The mutant toxin elicited high levels of neutralizing anti-ETA antibodies in mice, which protected against a challenge with 100 micrograms of authentic ETA (greater than 600 lethal doses). The mutant protein has the attributes of a toxoid and may be useful as a component of vaccines for individuals at risk for infection by P. aeruginosa.
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PMID:Toxoid of Pseudomonas aeruginosa exotoxin A generated by deletion of an active-site residue. 246 Apr 7

The enzymatic ADP-ribosyltransferase activity associated with the S1 subunit of pertussis toxin is considered to be responsible for its biological effects. Although pertussis toxin has no significant homology to other ADP-ribosylating toxins such as diphtheria toxin and Pseudomonas aeruginosa exotoxin A, the results presented in this paper show that, as for diphtheria toxin and exotoxin A, tryptophan and glutamic acid residues are essential for the enzymatic activities of pertussis toxin. Moreover, a structural motif can be identified around the critical glutamic acid residue. Chemical modification or site-directed deletion or replacement of Trp-26 abolishes ADP-ribosyltransferase and the associated NAD glycohydrolase activities. Both enzymatic activities are also abolished when Glu-129 is deleted or replaced by aspartic acid. Mutations at the Glu-106 position do not significantly reduce the enzymatic activities of the S1 subunit. The mutations do not affect the ability of the different S1 forms to be recognized by a variety of monoclonal antibodies, including neutralizing antibodies. Pertussis toxin containing a deletion or replacement of Trp-26, Glu-129, or both in the S1 subunit should thus be devoid of toxic activities without losing its reactivity with protective antibodies and, therefore, could be safely included in new generation vaccines against whooping cough.
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PMID:Identification of amino acid residues essential for the enzymatic activities of pertussis toxin. 247 88

Cysteines 265 and 287 of Pseudomonas aeruginosa exotoxin A (ETA) were substituted by serine, thereby eliminating a disulfide bridge within domain II, the putative membrane insertion-translocation domain. Purified mutant toxin was 80-fold less toxic for mouse L cells than was wild-type ETA while retaining the same specific activity in the ADP-ribosyltransferase reaction as did wild-type toxin. Binding of the nonionic detergent Triton X-114 by mutant ETA occurred at a slightly higher pH than did binding by wild-type ETA, suggesting that the mutant protein more readily undergoes a conformational change exposing hydrophobic regions. Data are presented supporting the notion that the mutant and wild-type toxins enter from the same intracellular compartment. The lower cytotoxicity of the mutant protein could be due to accelerated intracellular degradation or abortive, premature membrane insertion.
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PMID:Effects of eliminating a disulfide bridge within domain II of Pseudomonas aeruginosa exotoxin A. 249 39

Pseudomonas aeruginosa exotoxin A (ETA) is an ADP-ribosyltransferase which inactivates protein synthesis by covalently attaching the ADP-ribose portion of NAD+ onto eucaryotic elongation factor 2 (EF-2). A direct biochemical comparison has been made between ETA and a nonenzymatically active mutant toxin (CRM 66) using highly purified preparations of each protein. The loss of ADP-ribosyltransferase activity and subsequent cytotoxicity have been correlated with the presence of a tyrosine residue in place of a histidine at position 426 in CRM 66. In the native conformation, CRM 66 demonstrated a limited ability (by a factor or at least 100,000) to modify EF-2 covalently and lacked in vitro and in vivo cytotoxicity, yet CRM 66 appeared to be normal with respect to NAD+ binding. Upon activation with urea and dithiothreitol, CRM 66 lost ADP-ribosyltransferase activity entirely yet CRM 66 retained the ability to bind NAD+. Replacement of Tyr-426 with histidine in CRM 66 completely restored cytotoxicity and ADP-ribosyltransferase activity. These results support previous findings from this laboratory (Wozniak, D. J., Hsu, L.-Y., and Galloway, D. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8880-8884) which suggest that the His-426 residue of ETA is not involved in NAD+ binding but appears to be associated with the interaction between ETA and EF-2.
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PMID:Biochemical analysis of CRM 66. A nonfunctional Pseudomonas aeruginosa exotoxin A. 250 13


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