Importance of Conserved N-domain Residues Thr 441 , Glu 442 , Lys 515 , Arg 560 , and Leu 562 of Sarcoplasmic Reticulum Ca 2+ -ATPase for MgATP Binding and Subsequent Catalytic Steps: PLASTICITY OF THE NUCLEOTIDE-BINDING SITE

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dc.contributor.authorClausen, Johannes D
dc.contributor.authorMcIntosh, David B
dc.contributor.authorVilsen, Bente
dc.contributor.authorWoolley, David G
dc.contributor.authorAndersen, Jens Peter
dc.date.accessioned2021-10-08T07:20:48Z
dc.date.available2021-10-08T07:20:48Z
dc.date.issued2003
dc.description.abstractNine single mutations were introduced to amino acid residues Thr441, Glu442, Lys515, Arg560, Cys561, and Leu562 located in the nucleotide-binding domain of sarcoplasmic reticulum Ca2+-ATPase, and the functional consequences were studied in a direct nucleotide binding assay, as well as by steady-state and transient kinetic measurements of the overall and partial reactions of the transport cycle. Some partial reaction steps were also examined in mutants with alterations to Phe487, Arg489, and Lys492. The results implicate all these residues, except Cys561, in high affinity nucleotide binding at the substrate site. Mutations Thr441 --> Ala, Glu442 --> Ala, and Leu562 --> Phe were more detrimental to MgATP binding than to ATP binding, thus pointing to a role for these residues in the binding of Mg2+ or to a difference between the interactions with MgATP and ATP. Subsequent catalytic steps were also selectively affected by the mutations, showing the involvement of the nucleotide-binding domain in these reactions. Mutation of Arg560 inhibited phosphoryl transfer but enhanced the E1PCa2 --> E2P conformational transition, whereas mutations Thr441 --> Ala, Glu442 --> Ala, Lys492 --> Leu, and Lys515 --> Ala inhibited the E1PCa2 --> E2P transition. Hydrolysis of the E2P phosphoenzyme intermediate was enhanced in Glu442 --> Ala, Lys492 --> Leu, Lys515 --> Ala, and Arg560 --> Glu. None of the mutations affected the low affinity activation by nucleotide of the phosphoenzyme-processing steps, indicating that modulatory nucleotide interacts differently from substrate nucleotide. Mutation Glu442 --> Ala greatly enhanced reaction of Lys515 with fluorescein isothiocyanate, indicating that the two residues form a salt link in the native protein.
dc.identifier.apacitationClausen, J. D., McIntosh, D. B., Vilsen, B., Woolley, D. G., & Andersen, J. P. (2003). Importance of Conserved N-domain Residues Thr 441 , Glu 442 , Lys 515 , Arg 560 , and Leu 562 of Sarcoplasmic Reticulum Ca 2+ -ATPase for MgATP Binding and Subsequent Catalytic Steps: PLASTICITY OF THE NUCLEOTIDE-BINDING SITE. <i>The Journal of Biological Chemistry</i>, 278(22), 20245 - 20258. http://hdl.handle.net/11427/35007en_ZA
dc.identifier.chicagocitationClausen, Johannes D, David B McIntosh, Bente Vilsen, David G Woolley, and Jens Peter Andersen "Importance of Conserved N-domain Residues Thr 441 , Glu 442 , Lys 515 , Arg 560 , and Leu 562 of Sarcoplasmic Reticulum Ca 2+ -ATPase for MgATP Binding and Subsequent Catalytic Steps: PLASTICITY OF THE NUCLEOTIDE-BINDING SITE." <i>The Journal of Biological Chemistry</i> 278, 22. (2003): 20245 - 20258. http://hdl.handle.net/11427/35007en_ZA
dc.identifier.citationClausen, J.D., McIntosh, D.B., Vilsen, B., Woolley, D.G. & Andersen, J.P. 2003. Importance of Conserved N-domain Residues Thr 441 , Glu 442 , Lys 515 , Arg 560 , and Leu 562 of Sarcoplasmic Reticulum Ca 2+ -ATPase for MgATP Binding and Subsequent Catalytic Steps: PLASTICITY OF THE NUCLEOTIDE-BINDING SITE. <i>The Journal of Biological Chemistry.</i> 278(22):20245 - 20258. http://hdl.handle.net/11427/35007en_ZA
dc.identifier.issn0021-9258
dc.identifier.issn1083-351X
dc.identifier.ris TY - Journal Article AU - Clausen, Johannes D AU - McIntosh, David B AU - Vilsen, Bente AU - Woolley, David G AU - Andersen, Jens Peter AB - Nine single mutations were introduced to amino acid residues Thr441, Glu442, Lys515, Arg560, Cys561, and Leu562 located in the nucleotide-binding domain of sarcoplasmic reticulum Ca2+-ATPase, and the functional consequences were studied in a direct nucleotide binding assay, as well as by steady-state and transient kinetic measurements of the overall and partial reactions of the transport cycle. Some partial reaction steps were also examined in mutants with alterations to Phe487, Arg489, and Lys492. The results implicate all these residues, except Cys561, in high affinity nucleotide binding at the substrate site. Mutations Thr441 --> Ala, Glu442 --> Ala, and Leu562 --> Phe were more detrimental to MgATP binding than to ATP binding, thus pointing to a role for these residues in the binding of Mg2+ or to a difference between the interactions with MgATP and ATP. Subsequent catalytic steps were also selectively affected by the mutations, showing the involvement of the nucleotide-binding domain in these reactions. Mutation of Arg560 inhibited phosphoryl transfer but enhanced the E1PCa2 --> E2P conformational transition, whereas mutations Thr441 --> Ala, Glu442 --> Ala, Lys492 --> Leu, and Lys515 --> Ala inhibited the E1PCa2 --> E2P transition. Hydrolysis of the E2P phosphoenzyme intermediate was enhanced in Glu442 --> Ala, Lys492 --> Leu, Lys515 --> Ala, and Arg560 --> Glu. None of the mutations affected the low affinity activation by nucleotide of the phosphoenzyme-processing steps, indicating that modulatory nucleotide interacts differently from substrate nucleotide. Mutation Glu442 --> Ala greatly enhanced reaction of Lys515 with fluorescein isothiocyanate, indicating that the two residues form a salt link in the native protein. DA - 2003 DB - OpenUCT DP - University of Cape Town IS - 22 J1 - The Journal of Biological Chemistry LK - https://open.uct.ac.za PY - 2003 SM - 0021-9258 SM - 1083-351X T1 - Importance of Conserved N-domain Residues Thr 441 , Glu 442 , Lys 515 , Arg 560 , and Leu 562 of Sarcoplasmic Reticulum Ca 2+ -ATPase for MgATP Binding and Subsequent Catalytic Steps: PLASTICITY OF THE NUCLEOTIDE-BINDING SITE TI - Importance of Conserved N-domain Residues Thr 441 , Glu 442 , Lys 515 , Arg 560 , and Leu 562 of Sarcoplasmic Reticulum Ca 2+ -ATPase for MgATP Binding and Subsequent Catalytic Steps: PLASTICITY OF THE NUCLEOTIDE-BINDING SITE UR - http://hdl.handle.net/11427/35007 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/35007
dc.identifier.vancouvercitationClausen JD, McIntosh DB, Vilsen B, Woolley DG, Andersen JP. Importance of Conserved N-domain Residues Thr 441 , Glu 442 , Lys 515 , Arg 560 , and Leu 562 of Sarcoplasmic Reticulum Ca 2+ -ATPase for MgATP Binding and Subsequent Catalytic Steps: PLASTICITY OF THE NUCLEOTIDE-BINDING SITE. The Journal of Biological Chemistry. 2003;278(22):20245 - 20258. http://hdl.handle.net/11427/35007.en_ZA
dc.language.isoeng
dc.publisher.departmentDepartment of Clinical Laboratory Sciences
dc.publisher.facultyFaculty of Health Sciences
dc.sourceThe Journal of Biological Chemistry
dc.source.journalissue22
dc.source.journalvolume278
dc.source.pagination20245 - 20258
dc.source.urihttps://dx.doi.org/10.1074/jbc.M301122200
dc.subject.otherAdenosine Triphosphate
dc.subject.otherAmino Acid Sequence
dc.subject.otherAmino Acids
dc.subject.otherAnimals
dc.subject.otherBinding Sites
dc.subject.otherCalcium
dc.subject.otherCalcium-Transporting ATPases
dc.subject.otherCatalysis
dc.subject.otherConserved Sequence
dc.subject.otherHydrolysis
dc.subject.otherKinetics
dc.subject.otherModels, Molecular
dc.subject.otherMutagenesis
dc.subject.otherPhosphorylation
dc.subject.otherProtein Binding
dc.subject.otherRabbits
dc.subject.otherSarcoplasmic Reticulum
dc.titleImportance of Conserved N-domain Residues Thr 441 , Glu 442 , Lys 515 , Arg 560 , and Leu 562 of Sarcoplasmic Reticulum Ca 2+ -ATPase for MgATP Binding and Subsequent Catalytic Steps: PLASTICITY OF THE NUCLEOTIDE-BINDING SITE
dc.typeJournal Article
uct.type.publicationResearch
uct.type.resourceJournal Article
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