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Article: The conformation of coenzyme a bound to chloramphenicol acetyltransferase determined by transferred NOE experiments

TitleThe conformation of coenzyme a bound to chloramphenicol acetyltransferase determined by transferred NOE experiments
Authors
Issue Date1996
PublisherAcademic Press. The Journal's web site is located at http://www.elsevier.com/locate/jmb
Citation
Journal Of Molecular Biology, 1996, v. 262 n. 4, p. 543-558 How to Cite?
AbstractThe conformation of coenzyme A bound to chloramphenicol acetyltransferase has been studied in solution by NMR methods. Transferred nuclear Overhauser enhancement (NOE) and rotating frame NOE (ROE) experiments were used to determine the conformation of the bound coenzyme. Experiments were carried out at five mixing times and two temperatures, and with normal and perdeuterated enzyme, to ensure (1) that the fast exchange condition was satisfied and (2) that the results were not complicated by spin diffusion involving enzyme protons. The data were analysed using a general approach involving combined exchange and relaxation matrices. For the binary complex of coenzyme A (CoA) and enzyme, the conformation of CoA was calculated by using distance constraints derived from the intensities of 71 NOE and 33 ROE cross-peaks between coenzyme protons. The conformation of the adenosine moiety of CoA in the structure deduced by NMR is very close to that seen in the crystal structure of this complex, while the pantetheine moiety is clearly less extended. Essentially the same conformation was obtained whether or not the calculations included the protein (with appropriate intermolecular energy terms). The difference between the NMR and X-ray structures is interpreted in terms of the existence of two conformations of the CoA-enzyme complex. Support for this model comes from measurements of the coenzyme dissociation rate constant; NMR (lineshape analysis and transferred NOE experiments) gives estimates of k(off) ~ 3700 s-1 at 298 K and ~ 500 s-1 at 280 K, both significantly greater than estimates by fluorescence stopped-flow measurements. For the ternary complex of CoA, chloramphenicol and enzyme, 71 NOE cross-peaks between protons of coenzyme A and a further ten cross-peaks between protons of coenzyme A and chloramphenicol were measured. Starting with a model derived from the crystal structures of the two binary complexes (in the absence of crystallographic data for the ternary complex) the conformations and relative positions of the two ligands were refined using the distance constraints derived from these NOEs. The conformation of the adenosine part of CoA is the same as in the binary complex, while the pantetheine arm is more extended and approaches close to the bound chloramphenicol molecule. The model of the ternary complex is discussed in terms of the information available on the mechanism of the enzyme.
Persistent Identifierhttp://hdl.handle.net/10722/157274
ISSN
2015 Impact Factor: 4.517
2015 SCImago Journal Rankings: 3.002
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorBarsukov, ILen_US
dc.contributor.authorLian, LYen_US
dc.contributor.authorEllis, Jen_US
dc.contributor.authorSze, KHen_US
dc.contributor.authorShaw, WVen_US
dc.contributor.authorRoberts, GCKen_US
dc.date.accessioned2012-08-08T08:48:34Z-
dc.date.available2012-08-08T08:48:34Z-
dc.date.issued1996en_US
dc.identifier.citationJournal Of Molecular Biology, 1996, v. 262 n. 4, p. 543-558en_US
dc.identifier.issn0022-2836en_US
dc.identifier.urihttp://hdl.handle.net/10722/157274-
dc.description.abstractThe conformation of coenzyme A bound to chloramphenicol acetyltransferase has been studied in solution by NMR methods. Transferred nuclear Overhauser enhancement (NOE) and rotating frame NOE (ROE) experiments were used to determine the conformation of the bound coenzyme. Experiments were carried out at five mixing times and two temperatures, and with normal and perdeuterated enzyme, to ensure (1) that the fast exchange condition was satisfied and (2) that the results were not complicated by spin diffusion involving enzyme protons. The data were analysed using a general approach involving combined exchange and relaxation matrices. For the binary complex of coenzyme A (CoA) and enzyme, the conformation of CoA was calculated by using distance constraints derived from the intensities of 71 NOE and 33 ROE cross-peaks between coenzyme protons. The conformation of the adenosine moiety of CoA in the structure deduced by NMR is very close to that seen in the crystal structure of this complex, while the pantetheine moiety is clearly less extended. Essentially the same conformation was obtained whether or not the calculations included the protein (with appropriate intermolecular energy terms). The difference between the NMR and X-ray structures is interpreted in terms of the existence of two conformations of the CoA-enzyme complex. Support for this model comes from measurements of the coenzyme dissociation rate constant; NMR (lineshape analysis and transferred NOE experiments) gives estimates of k(off) ~ 3700 s-1 at 298 K and ~ 500 s-1 at 280 K, both significantly greater than estimates by fluorescence stopped-flow measurements. For the ternary complex of CoA, chloramphenicol and enzyme, 71 NOE cross-peaks between protons of coenzyme A and a further ten cross-peaks between protons of coenzyme A and chloramphenicol were measured. Starting with a model derived from the crystal structures of the two binary complexes (in the absence of crystallographic data for the ternary complex) the conformations and relative positions of the two ligands were refined using the distance constraints derived from these NOEs. The conformation of the adenosine part of CoA is the same as in the binary complex, while the pantetheine arm is more extended and approaches close to the bound chloramphenicol molecule. The model of the ternary complex is discussed in terms of the information available on the mechanism of the enzyme.en_US
dc.languageengen_US
dc.publisherAcademic Press. The Journal's web site is located at http://www.elsevier.com/locate/jmben_US
dc.relation.ispartofJournal of Molecular Biologyen_US
dc.subject.meshChloramphenicol O-Acetyltransferase - Chemistry - Metabolismen_US
dc.subject.meshCoenzyme A - Chemistry - Metabolismen_US
dc.subject.meshCrystallography, X-Rayen_US
dc.subject.meshMagnetic Resonance Spectroscopyen_US
dc.subject.meshProtein Conformationen_US
dc.titleThe conformation of coenzyme a bound to chloramphenicol acetyltransferase determined by transferred NOE experimentsen_US
dc.typeArticleen_US
dc.identifier.emailSze, KH:khsze@hku.hken_US
dc.identifier.authoritySze, KH=rp00785en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1006/jmbi.1996.0534en_US
dc.identifier.pmid8893862-
dc.identifier.scopuseid_2-s2.0-0030568979en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0030568979&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume262en_US
dc.identifier.issue4en_US
dc.identifier.spage543en_US
dc.identifier.epage558en_US
dc.identifier.isiWOS:A1996VK74900013-
dc.publisher.placeUnited Kingdomen_US
dc.identifier.scopusauthoridBarsukov, IL=35586964900en_US
dc.identifier.scopusauthoridLian, LY=7005156195en_US
dc.identifier.scopusauthoridEllis, J=7402715294en_US
dc.identifier.scopusauthoridSze, KH=7006735061en_US
dc.identifier.scopusauthoridShaw, WV=7202632977en_US
dc.identifier.scopusauthoridRoberts, GCK=7403400348en_US

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