Abstract
ATP binds to sarcoplasmic reticulum Ca(2+)-ATPase both in a phosphorylating (catalytic) mode and in a nonphosphorylating (modulatory) mode, the latter leading to acceleration of phosphoenzyme turnover (Ca(2)E(1)P --> E(2)P and E(2)P --> E(2) reactions) and Ca(2+) binding (E(2) --> Ca(2)E(1)). In some of the Ca(2+)-ATPase crystal structures, Arg(678) and Glu(439) seem to be involved in the binding of nucleotide or an associated Mg(2+) ion. We have replaced Arg(678), Glu(439), and Gly(438) with alanine to examine their importance for the enzyme cycle and the modulatory effects of ATP and MgATP. The results point to the key role of Arg(678) in nucleotide binding and to the importance of interdomain bonds Glu(439)-Ser(186) and Arg(678)-Asp(203) in stabilizing the E(2)P and E(2) intermediates, respectively. Mutation of Arg(678) had conspicuous effects on ATP/MgATP binding to the E(1) form and ADP binding to Ca(2)E(1)P, as well as ATP/MgATP binding in modulatory modes to E(2)P and E(2), whereas the effects on ATP/MgATP acceleration of the Ca(2)E(1)P --> E(2)P transition were small, suggesting that the nucleotide that accelerates Ca(2)E(1)P --> E(2)P binds differently from that modulating the E(2)P --> E(2) and E(2) --> Ca(2)E(1) reactions. Mutation of Glu(439) hardly affected nucleotide binding to E(1), Ca(2)E(1)P, and E(2), but it led to disruption of the modulatory effect of ATP on E(2)P --> E(2) and acceleration of the latter reaction, indicating that ATP normally modulates E(2)P --> E(2) by interfering with the interaction between Glu(439) and Ser(186). Gly(438) seems to be important for this interaction as well as for nucleotide binding, probably because of its role in formation of the helix containing Glu(439) and Thr(441).
Highlights
20686 JOURNAL OF BIOLOGICAL CHEMISTRY ples ATP hydrolysis with Ca2ϩ translocation against a concentration gradient by means of a reaction cycle (Scheme 1) in which the ATPase enzyme is transiently phosphorylated at a conserved aspartic acid residue and undergoes major conformational transitions between Ca2E1/Ca2E1P and E2/E2P forms [2, 3]
In the Ca2E1 and Ca2E1P conformations, the catalytic ATPbinding site is made up by residues in the N- and P-domains, and during the Ca2E1P 3 E2P transition the departing ADP molecule is replaced by the TGES loop of the A-domain, which subsequently assists in catalysis of E2P dephosphorylation [8, 9, 12]
A subject of much controversy is the question whether the phosphorylating and modulatory ATP molecules are at the same locus, exhibiting variable affinity during the transport cycle depending on conformational state, or whether a separate low affinity allosteric site exists on the same Ca2ϩ-ATPase polypeptide chain (18, 22, 24 –28)
Summary
Distance nucleotide/Mg-amino acid residue (closest distance and other relevant distances). E21⁄7CPA1⁄7Mga1⁄7ADP (PDB code 2OA0; see Ref. 11) a Mg2ϩ or Ca2ϩ ion present at the “canonical” Mg2ϩ site 1. B Mg2ϩ present at site 2 close to the ␣-phosphate of the nucleotide. Function of the adjacent glycine residue Gly438, which appears too far away for direct interaction with the nucleotide or its associated Mg2ϩ (cf Table 1 and Fig. 1), but is well conserved, was examined by replacement with alanine. All the partial reactions of the Ca2ϩ-ATPase reaction cycle indicated in Scheme 1 and their ATP dependences were analyzed in the mutants to address the effects of the mutations on the conformational changes of the enzyme as well as the interaction with ATP in catalytic as well as modulatory modes
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