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

Nine single mutations were introduced to amino acid residues Thr(441), Glu(442), Lys(515), Arg(560), Cys(561), and Leu(562) 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 Phe(487), Arg(489), and Lys(492). The results implicate all these residues, except Cys(561), in high affinity nucleotide binding at the substrate site. Mutations Thr(441)-->Ala, Glu(442)-->Ala, and Leu(562) --> 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 Thr(441) --> Ala, Glu(442) --> Ala, Lys(492) --> Leu, and Lys(515) --> Ala inhibited the E1PCa2 --> E2P transition. Hydrolysis of the E2P phosphoenzyme intermediate was enhanced in Glu(442) --> Ala, Lys(492) --> Leu, Lys(515) --> Ala, and Arg(560)-->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 Glu(442) --> Ala greatly enhanced reaction of Lys(515) with fluorescein isothiocyanate, indicating that the two residues form a salt link in the native protein.