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)

Exoenzyme S of Pseudomonas aeruginosa (ExoS) is a member of the family of bacterial ADP-ribosylating exotoxins (bAREs). Site-directed mutagenesis of glutamic acids within the catalytic domain of ExoS (termed delta N222) allowed the identification of the preferential inactivation of ADP-ribosyltransferase activity by alanine substitution of E381. The specific activity of E381A mutant was 0.02% of wild-type delta N222. Delta N222(E381A) retained the requirement of factor activating exoenzyme S (FAS) activation for the expression of ADP-ribosyltransferase activity. In contrast, E387A, E399A, and E414A mutants possessed ADP-ribosyltransferase activity similar to that of wild-type delta N222. Kinetic evaluation of E381A and two other mutants, E381D and E381S, showed that their primary defect was a lower kcat in the ADP-ribosylation of soybean trypsin inhibitor (SBTI). The Km for NAD and SBTI and activation by FAS varied 2- and 10-fold relative to delta N222. In addition, the E381 mutants possessed identical protease patterns during thrombin and trypsin digestion as delta N222, which indicated that E381 mutants had retained their overall conformation. Together, these data identify E381 as contributing to the catalytic activity of exoenzyme S.
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PMID:Identification of glutamic acid 381 as a candidate active site residue of Pseudomonas aeruginosa exoenzyme S. 861 82

Earlier studies reported that Pseudomonas aeruginosa exoenzyme S (ExoS) possessed an absolute requirement for the eukaryotic protein factor activating exoenzyme S (FAS) for expressing ADP-ribosyltransferase activity. During the characterization of a serum-derived FAS-like activity, we observed the ability of a catalytic deletion peptide of ExoS (DeltaN222) to ADP-ribosylate target proteins in the absence of FAS. Characterization of the activation of DeltaN222 by FAS provided an opportunity to gain insight into the mechanism of ExoS activation by FAS. Under standard enzyme assay conditions, the initial rate of FAS-independent ADP-ribosyltransferase activity of DeltaN222 was not linear with time and rapidly approached zero. Dilution into high-ionic strength buffers stabilized DeltaN222 so it could express FAS-independent ADP-ribosyltransferase activity at a linear rate. This stabilization was a general salt effect, since dilution into a 1.0 M solution of either NaCH3COOH, NaCl, or KCl stabilized the ADP-ribosyltransferase activity of DeltaN222. Kinetic analysis in a high-ionic strength buffer showed that FAS enhanced the catalytic activity of DeltaN222 by increasing the affinity for NAD and stimulating the turnover rate. Velocity experiments indicated that the stabilization of DeltaN222 by high salt was not functionally identical to stabilization by FAS. Together, these data implicate a dual role for FAS in the allosteric activation of ExoS, involving both substrate binding and catalysis.
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PMID:Expression of FAS-independent ADP-ribosyltransferase activity by a catalytic deletion peptide of Pseudomonas aeruginosa exoenzyme S. 1023 37