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)

Glutamine synthetase from the photosynthetic bacterium Rhodospirillum rubrum is the target of both ATP- and NAD-dependent modification. Incubation of R. rubrum cell supernatant with [alpha-32P]NAD results in the labeling of glutamine synthetase and two other unidentified proteins. Dinitrogenase reductase ADP-ribosyltransferase does not appear to be responsible for the modification of glutamine synthetase or the unidentified proteins. The [alpha-32P]ATP- and [alpha-32P] NAD-dependent modifications of R. rubrum glutamine synthetase appear to be exclusive and the two forms of modified glutamine synthetase are separable on two-dimensional gels. Loss of enzymatic activity by glutamine synthetase did not correlate with [alpha-32P]NAD labeling. This is in contrast to inactivation by nonphysiological ADP-ribosylation of other glutamine synthetases by an NAD:arginine ADP-ribosyltransferase from turkey erythrocytes (Moss, J., Watkins, P.A., Stanley, S.J., Purnell, M.R., and Kidwell, W.R. (1984) J. Biol. Chem. 259, 5100-5104). A 32P-labeled protein spot comigrates with the NAD-treated glutamine synthetase spot when glutamine synthetase purified from H3 32PO4-grown cells is analyzed on two-dimensional gels. The adenylylation site of R. rubrum glutamine synthetase has been determined to be Leu-(Asp)-Tyr-Leu-Pro-Pro-Glu-Glu-Leu-Met; the tyrosine residue is the site of modification.
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PMID:ATP-dependent and NAD-dependent modification of glutamine synthetase from Rhodospirillum rubrum in vitro. 197 53

Glutamic acid-148, an active-site residue of diphtheria toxin identified by photoaffinity labeling with NAD, was replaced with aspartic acid, glutamine, or serine by directed mutagenesis of the F2 fragment of the toxin gene. Wild-type and mutant F2 proteins were synthesized in Escherichia coli, and the corresponding enzymic fragment A moieties (DTA) were derived, purified, and characterized. The Glu----Asp (E148D), Glu----Gln (E148Q), and Glu----Ser (E148S) mutations caused reductions in NAD:EF-2 ADP-ribosyltransferase activity of ca. 100-, 250-, and 300-fold, respectively, while causing only minimal changes in substrate affinity. The effects of the mutations on NAD-glycohydrolase activity were considerably different; only a 10-fold reduction in activity was observed for E148S, and the E148D and E148Q mutants actually exhibited a small but reproducible increase in NAD-glycohydrolytic activity. Photolabeling by nicotinamide-radiolabeled NAD was diminished ca. 8-fold in the E148D mutant and was undetectable in the other mutants. The results confirm that Glu-148 plays a crucial role in the ADP-ribosylation of EF-2 and imply an important function for the side-chain carboxyl group in catalysis. The carboxyl group is also important for photochemical labeling by NAD but not for NAD-glycohydrolase activity. The pH dependence of the catalytic parameters for the ADP-ribosyltransferase reaction revealed a group in DTA-wt that titrates with an apparent pKa of 6.2-6.3 and is in the protonated state in the rate-determining step.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine. 198 Feb 8

The mechanism for "NH4+ switch-off/on" of nitrogenase activity in Azospirillum brasilense and A. lipoferum was investigated. A correlation was established between the in vivo regulation of nitrogenase activity by NH4Cl or glutamine and the reversible covalent modification of dinitrogenase reductase. Dinitrogenase reductase ADP-ribosyltransferase (DRAT) activity was detected in extracts of A. brasilense with NAD as the donor molecule. Dinitrogenase reductase-activating glycohydrolase (DRAG) activity was present in extracts of both A. brasilense and A. lipoferum. The DRAG activity in A. lipoferum was membrane associated, and it catalyzed the activation of inactive nitrogenase (by covalent modification of dinitrogenase reductase) from both A. lipoferum and Rhodospirillum rubrum. A region homologous to R. rubrum draT and draG was identified in the genomic DNA of A. brasilense as a 12-kilobase EcoRI fragment and in A. lipoferum as a 7-kilobase EcoRI fragment. It is concluded that a posttranslational regulatory system for nitrogenase activity is present in A. brasilense and A. lipoferum and that it operates via ADP-ribosylation of dinitrogenase reductase as it does in R. rubrum.
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PMID:Posttranslational regulatory system for nitrogenase activity in Azospirillum spp. 250 94

Sulfhydryl-alkylating reagents are known to inactivate the NAD glycohydrolase and ADP-ribosyltransferase activities of the S1 subunit of pertussis toxin, a protein which contains two cysteines at positions 41 and 200. It has been proposed that NAD can retard alkylation of one of the two cysteines of this protein (Kaslow, H.R., and Lesikar, D.D. (1987) Biochemistry 26, 4397-4402). We now report that NAD retards the ability of these alkylating reagents to inactivate the S1 subunit. In order to determine which cysteine is protected by NAD, we used site-directed mutagenesis to construct analogs of the toxin with serines at positions 41 and/or 200. Sulfhydryl-alkylating reagents reduced the ADP-ribosyltransferase activity of the analog with a single cysteine at position 41; NAD retarded this inactivation. In contrast, sulfhydryl-alkylating reagents did not inactivate analogs with serine at position 41. An analog with alanine at position 41 possessed substantial ADP-ribosyltransferase activity. We conclude that alkylation of cysteine 41, and not cysteine 200, inactivates the S1 subunit of pertussis toxin, but that the sulfhydryl group of cysteine 41 is not essential for the ADP-ribosyltransferase activity of the toxin. These results suggest that the region near cysteine 41 contributes to features of the S1 subunit important for ADP-ribosyltransferase activity. Using site-directed mutagenesis, we found that changing aspartate 34 to asparagine, arginine 39 to lysine, and glutamine 42 to glutamate had little effect on ADP-ribosyltransferase activity. However, substituting an asparagine for the histidine at position 35 markedly decreased, but did not eliminate, ADP-ribosyltransferase activity. Chou-Fasman analysis predicted no significant modifications in secondary structure of the S1 peptide with the change of histidine 35 to asparagine. Thus, histidine 35 may interact with a substrate of the S1 subunit without being essential for catalysis.
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PMID:Alkylation of cysteine 41, but not cysteine 200, decreases the ADP-ribosyltransferase activity of the S1 subunit of pertussis toxin. 270 95

Clostridium limosum ADP-ribosyltransferase modifies low molecular mass GTP-binding proteins of the Rho subtype family. Here we cloned and sequenced the gene of the transferase and expressed it in Escherichia coli. The gene encodes a protein of 250 amino acids (M(r) = 27,840), with a putative signal peptide of 45 amino acids, that shows about 60-65% identity with C3 transferases from Clostridium botulinum. The mature C. limosum transferase was expressed as a maltose-binding fusion protein in E. coli and purified to apparent homogeneity. To study the functional role of Glu174 of C. limosum transferase, which was recently photoaffinity-labeled with [carbonyl-14C]NAD [Jung, M., et al. (1993) J. Biol. Chem. 268, 23215-23218], two mutants E174D and E174Q were constructed by a polymerase chain reaction-based system. The E174D and E174Q mutants showed a dramatic decrease in kcat, but no major changes in Km,NAD. Furthermore, replacement of Glu174 by aspartic acid and glutamine largely reduced and completely blocked UV-induced incorporation of [carbonyl-14C]NAD into the transferase. The data indicate that Glu174 is an active site residue of C. limosum transferase.
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PMID:Active site mutation of the C3-like ADP-ribosyltransferase from Clostridium limosum--analysis of glutamic acid 174. 855 86

The actin ADP-ribosylating Clostridium botulinum C2 toxin is a binary toxin composed of the binding component C2II and the enzyme component C2I. C2I ADP-ribosylates G-actin at arginine 177, resulting in the depolymerization of the actin cytoskeleton. Here, we studied the structure-function relationship of C2I by site-directed mutagenesis. Exchange of Glu389 to glutamine caused the complete loss of ADP-ribosyltransferase and NAD-glycohydrolase activities of C2I. In contrast, exchange of Glu387 to glutamine blocked ADP-ribosyltransferase but not NAD-glycohydrolase activity. Whereas photoaffinity labeling of the double mutant E387Q/E389Q C2I with [carbonyl-14C]NAD was blocked, labeling of the single C2I mutants was reduced (E389Q) or not changed (E387Q). Exchange of the STS motif (amino acid residues 348-350) of C2I caused a decrease in transferase activity by more than 99 (S348A) and 90% (T349V), or did not affect activity (S350A). Exchange of Arg299 and Arg300 to lysine reduced transferase activity to <0.1 and approximately 35% of wild-type activity. The data indicate that the amino acid residues Glu389, Glu387, Ser348, and Arg299, which are conserved in various prokaryotic and eukaryotic arginine-modifying ADP-ribosyltransferases, are essential for ADP-ribosyltransferase activity of the enzyme component of C. botulinum C2 toxin.
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PMID:Characterization of the catalytic site of the ADP-ribosyltransferase Clostridium botulinum C2 toxin by site-directed mutagenesis. 979 57

The iota(a) component (i(a)) of Clostridium perfringens ADP ribosylates nonmuscle beta/gamma actin and skeletal muscle alpha-actin. Replacement of Arg-295 in i(a) with alanine led to a complete loss of NAD(+)-glycohydrolase (NADase) and ADP-ribosyltransferase (ARTase); that of the residue with lysine caused a drastic reduction in NADase and ARTase activities (<0.1% of the wild-type activities) but did not completely diminish them. Substitution of alanine for Glu-378 and Glu-380 caused a complete loss of NADase and ARTase. However, exchange of Glu-378 to aspartic acid or glutamine resulted in little effect on NADase activity but a drastic reduction in ARTase activity (<0.1% of the wild-type activity). Exchange of Glu-380 to aspartic acid caused a drastic reduction in NADase and ARTase activities (<0.1% of the wild-type activities) but did not completely diminish them; that of the residue to glutamine caused a complete loss of ARTase activity. Replacement of Ser-338 with alanine resulted in 0.7 to 2.3% wild-type activities, and that of Ser-340 and Thr-339 caused a reduction in these activities of 5 to 30% wild-type activities. The kinetic analysis showed that Arg-295 and Ser-338 also play an important role in the binding of NAD(+) to i(a), that Arg-295, Glu-380, and Ser-338 play a crucial role in the catalytic rate of NADase activity, and that these three amino acid residues and Glu-378 are essential for ARTase activity. The effect of amino acid replacement in i(a) on ARTase activity was similar to that on lethal and cytotoxic activities, suggesting that lethal and cytotoxic activities in i(a) are dependent on ARTase activity.
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PMID:Characterization of the enzymatic component of Clostridium perfringens iota-toxin. 1073 50

Certain strains of Clostridium difficile produce the ADP-ribosyltransferase CDT, which is a binary actin ADP-ribosylating toxin. The toxin consists of the binding component CDTb, which mediates receptor binding and cellular uptake, and the enzyme component CDTa. Here we studied the enzyme component (CDTa) of the toxin using the binding component of Clostridium perfringens iota toxin (Ib), which is interchangeable with CDTb as a transport component. Ib was used because CDTb was not expressed as a recombinant protein in Escherichia coli. Similar to iota toxin, CDTa ADP-ribosylates nonmuscle and skeletal muscle actin. The N-terminal part of CDTa (CDTa1-240) competes with full-length CDTa for binding to the iota toxin binding component. The C-terminal part (CDTa244-263) harbors the enzyme activity but was much less active than the full-length CDTa. Changes of Glu428 and Glu430 to glutamine, Ser388 to alanine, and Arg345 to lysine blocked ADP-ribosyltransferase activity. Comparison of CDTa with C. perfringens iota toxin and Clostridium botulinum C2 toxin revealed full enzyme activity of the fragment Ia208-413 but loss of activity of several N-terminally deleted C2I proteins including C2I103-431, C2I190-431, and C2I30-431. The data indicate that CDTa belongs to the iota toxin subfamily of binary actin ADP-ribosylating toxins with respect to interaction with the binding component and substrate specificity. It shares typical conserved amino acid residues with iota toxin and C2 toxin that are suggested to be involved in NAD-binding and/or catalytic activity. The enzyme components of CDT, iota toxin, and C2 toxin differ with respect to the minimal structural requirement for full enzyme activity.
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PMID:Characterization of the enzymatic component of the ADP-ribosyltransferase toxin CDTa from Clostridium difficile. 1155 37

Exoenzyme C3stau2 from Staphylococcus aureus is a new member of the family of C3-like ADP-ribosyltransferases that ADP-ribosylates RhoA, -B, and -C. Additionally, it modifies RhoE and Rnd3. Here we report on studies of the structure-function relationship of recombinant C3stau2 by site-directed mutagenesis. Exchange of Glu(180) with leucine caused a complete loss of both ADP-ribosyltransferase and NAD glycohydrolase activity. By contrast, exchange of the glutamine residue two positions upstream (Gln(178)) with lysine blocked ADP-ribosyltransferase activity without major changes in NAD glycohydrolase activity. NAD and substrate binding of this mutant protein was comparable to that of the recombinant wild type. Exchange of amino acid Tyr(175), which is part of the recently described "ADP-ribosylating toxin turn-turn" (ARTT) motif [Han, S., Arvai, A. S., Clancy, S. B., and Tainer, J. A. (2001) J. Mol.Biol. 305, 95-107], with alanine, lysine, or threonine caused a loss of or a decrease in ADP-ribosyltransferase activity but an increase in NAD glycohydrolase activity. Recombinant C3stau2 Tyr175Ala and Tyr175Lys were not precipitated by matrix-bound Rho, supporting a role of Tyr(175) in protein substrate recognition. Exchange of Arg(48) and/or Arg(85) resulted in a 100-fold reduced transferase activity, while the recombinant C3stau2 double mutant R48K/R85K was totally inactive. The data indicate that amino acid residues Arg(48), Arg(85), Tyr(175), Gln(178), and Glu(180) are essential for ADP-ribosyltransferase activity of recombinant C3stau2 and support the role of the ARTT motif in substrate recognition of RhoA by C3-like ADP-ribosyltransferases.
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PMID:Structure-function analysis of the Rho-ADP-ribosylating exoenzyme C3stau2 from Staphylococcus aureus. 1181 47

Nucleated cells are more resistant to complement-mediated cell death than anucleated cells such as erythrocytes. There are few reports concerning the metabolic response of nucleated cells subjected to sub-lethal complement attack. It is possible that the rate of utilization of specific metabolic fuels by the cell is increased to enhance cell defence. We have measured the maximum activity of hexokinase, citrate synthase, glucose 6-phosphate dehydrogenase and glutaminase in rat mesenteric lymphocytes exposed to sub-lethal concentrations of activated complement (present in zymosan-activated serum, ZAS). These enzymes were carefully selected as they indicate changes of flux in glycolysis, TCA cycle, pentose phosphate pathway and glutaminolysis, respectively. The only enzyme activity to change on exposure of lymphocytes to ZAS was glutaminase, which was enhanced approximately by two-fold. Although rates of both glutamine and glucose utilization were enhanced by exposure to ZAS, only the rate of oxidation of glutamine was increased. Complement kills anucleated cells by simple osmotic lysis. However, it is likely that some nucleated cells will display characteristics of an ordered death mechanism and we have demonstrated that the concentration of lymphocyte ATP is dramatically decreased by activated complement. Nevertheless, the extent of cell death could be significantly reduced by the addition of inhibitors of the nuclear enzyme poly (ADP-ribose) polymerase (PARP). We conclude that glutamine metabolism is not only important for lymphocyte proliferative responses but is also important for cell defence from sub-lethal concentrations of activated complement. The rapid rate of complement-induced lymphocyte death reported here is suggested to be a consequence of over-activation of the nuclear enzyme PARP and ATP depletion.
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PMID:Sub-lethal concentrations of activated complement increase rat lymphocyte glutamine utilization and oxidation while lethal concentrations cause death by a mechanism involving ATP depletion. 1212 93


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