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J. Biol. Chem., Vol. 278, Issue 22, 20245-20258, May 30, 2003

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Importance of Conserved N-domain Residues Thr⁴⁴¹, Glu⁴⁴², Lys⁵¹⁵, Arg⁵⁶⁰, and Leu⁵⁶² of Sarcoplasmic Reticulum Ca²⁺-ATPase for MgATP Binding and Subsequent Catalytic Steps

PLASTICITY OF THE NUCLEOTIDE-BINDING SITE^*

Johannes D. Clausen , David B. McIntosh  ¶, Bente Vilsen , David G. Woolley  and Jens Peter Andersen  ||

From the Department of Physiology, University of Aarhus, DK-8000 Aarhus C, Denmark and the Division of Chemical Pathology, Department of Clinical Laboratory Sciences, Faculty of Health Sciences, University of Cape Town, Cape Town 7925, South Africa

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.

Received for publication, February 3, 2003

^* This work was supported in part by grants from the Danish Medical Research Council, the Novo Nordisk Foundation, Denmark, the Lundbeck Foundation, Denmark, the Research Foundation of Aarhus University (to J. P. A. and B. V.), and the National Research Foundation of South Africa (to D. B. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^{¶ ||} ¶ To whom correspondence may be addressed: Division of Chemical Pathology, Dept. of Clinical Laboratory Sciences, Faculty of Health Sciences, University of Cape Town, Cape Town 7925, South Africa. Fax: 27-21-4488150; E-mail: davidmci{at}chempath.uct.ac.za.|| To whom correspondence may be addressed: Department of Physiology, University of Aarhus, Ole Worms Allé 160, DK-8000 Aarhus C, Denmark. Fax: 45-86129065; E-mail: jpa{at}fi.au.dk.

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