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dc.contributor.authorCheng, Huien_US
dc.date.accessioned2014-03-14T20:35:37Z
dc.date.available2014-03-14T20:35:37Z
dc.date.issued2003-05-01en_US
dc.identifier.otheretd-05082003-122622en_US
dc.identifier.urihttp://hdl.handle.net/10919/32364
dc.description.abstractPspE, encoded by the last gene of the phage shock protein operon, is one of the nine proteins of Escherichia coli that contain a rhodanese homology domain. PspE is synthesized as a precursor with a 19-amino acid signal sequence and secreted to the periplasm. Mature PspE is the smallest rhodanese of E. coli (85 amino acids) and catalyzes the transfer of sulfur from thiosulfate to cyanide forming thiocyanate and sulfite. Cation exchange chromatography of a freeze-thaw extract of a PspE-overexpressing strain yielded two major peaks of active, homogeneous PspE. The two peaks contained two forms of PspE (PspE1 and PspE2) of distinct size and/or charge that were distinguished by native polyacrylamide gel electrophoresis and gel chromatography. PspE2 was converted to the more compact PspE1 by treatment with thiosulfate, which suggested that PspE1 is the persulfide form. One equivalent of cyanizable sulfur was associated with PspE1, with much less present in PspE2. Consistent with the conclusion that the single active site cysteine of PspE1 contains a persulfide sulfur was the observation that this form was much more tolerant to chemical inactivation by thiol-specific modifying reagent DTNB (5,5â -dithiobis(2-nitrobenzoic acid)). Rhodanese activity was subject to inhibition by anions (sulfite, sulfate, chloride, phosphate and arsenate), suggesting PspE has a cationic site for substrate binding. Kinetic analysis revealed that PspE employs a double-displacement mechanism, as is the case for other rhodaneses. The Kms for SSO32- and CN- were 3.0 and 43 mM, respectively. PspE exhibited a kcat of 72 s-1. To aid in understanding the physiological role of PspE, a strain with a pspE gene disruption was constructed. Comparison of rhodanese activity in extracts of wild-type and mutant strains revealed that PspE is a major contributor of rhodanese activity in E. coli. The pspE mutant displayed no obvious growth defect or auxotrophies, and was capable of molybdopterin biosynthesis, indicating that pspE is not essential for production of sulfur-containing amino acid or cofactors. Growth of wild-type and mutant strains deficient in pspE and other sulfurtransferase paralogs in medium with cyanide or cadmium was compared. The results indicated that neither PspE nor other E. coli rhodanese paralogs play roles in cyanide or cadmium detoxification. The physiological role of PspE remains to be determined.en_US
dc.publisherVirginia Techen_US
dc.relation.haspartthesis0521.pdfen_US
dc.rightsI hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Virginia Tech or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.en_US
dc.subjectsulfurtransferaseen_US
dc.subjectPspEen_US
dc.subjectEscherichia colien_US
dc.subjectrhodaneseen_US
dc.titleCharacterization of PspE, a Secreted Sulfurtransferase of Escherichia colien_US
dc.typeThesisen_US
dc.contributor.departmentBiochemistryen_US
dc.description.degreeMaster of Scienceen_US
thesis.degree.nameMaster of Scienceen_US
thesis.degree.levelmastersen_US
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen_US
thesis.degree.disciplineBiochemistryen_US
dc.contributor.committeechairLarson, Timothy J.en_US
dc.contributor.committeememberPopham, David L.en_US
dc.contributor.committeememberDean, Dennis R.en_US
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-05082003-122622/en_US
dc.date.sdate2003-05-08en_US
dc.date.rdate2012-06-22
dc.date.adate2003-05-22en_US


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