The sarcoplasmic reticulum (SR)1 Ca 2ϩ -ATPase belongs to a family of cation transport P-type ATPases that are phosphorylated by ATP on an aspartyl residue during the catalytic cycle. Ca 2ϩ translocation occurs in the first part of the cycle and is activated by Ca 2ϩ and MgATP binding to separate high affinity sites leading to phosphorylation and Ca 2ϩ occlusion within the protein (1-5). Phosphorylation appears to be facilitated by an ATP-induced conformational change which may align enzymatic groups in the transition state (6). A nucleotide-dependent conformational change in the presence of Ca 2ϩ is recorded by probes attached to Cys 674 (7-10) and is seen also by the increased reactivity of the latter (8, 11) and the exposure of a critical, reducible disulfide (12). A further conformational change of the phosphoenzyme permits Ca 2ϩ release to the lumen (13, 14). The cycle is completed with dephosphorylation and the associated transport of H ϩ counterions (15). The pump cycle is regulated by ATP. Ca 2ϩ binding (16 -20), Ca 2ϩ release to the lumen and the E1P to E2P transition (13, 21-23), and dephosphorylation (24 -27) are accelerated by ATP in different concentration ranges. These modulations result in a complex dependence of ATP hydrolysis on ATP concentration (1, 23, 28, 29). The regulatory ATP binding site has been the subject of intense study over many years, and there is increasing evidence, mainly from results obtained with probes covalently attached at the catalytic site (27, 30 -32), that at least part of the effect is due to ATP rebinding at or close to the catalytic site following phosphoenzyme formation and ADP dissociation.The identification and role of residues complexing ATP, in either a catalytic or a regulatory mode, are being elucidated by a variety of approaches. Chemical modification and photoaffinity labeling have implicated Lys 492 (31,(33)(34)(35)(36)(37) ) appear to be involved in binding ATP in a regulatory capacity following phosphorylation. However, some doubt has been cast on the role of Lys 492 in ATP binding because derivatization with 7-amino-4-methylcoumarin-3-acetic acid succinimidyl ester apparently has no effect on ATPase activity (37). The function of Lys 492 may be complex, because derivatization with TNP-8N 3 -ATP partially uncoupled Ca 2ϩ transport from hydrolysis of the tethered nucleotide (32) and cross-linking Lys 492 to Arg 678 with glutaraldehyde permitted ATP-dependent Ca 2ϩ occlusion but completely blocked Ca 2ϩ release to the lumen (14).Site-directed mutagenesis has been used to probe several conserved amino acids and segments for involvement in ATP binding. Defective ATP binding following mutation of Lys

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-and pH-dependent equilibrium (7) of several (E1/E2) conformational states (Scheme 1) that appear to interact differently with ATP. E2H 3 7 E2H 7 E1 7 E1Ca 7 E1Ca 2 SCHEME 1 Although in the presence of Mg 2ϩ all of the states indicated in Scheme 1 exhibit rather high affinities for ATP (K D in the range 0.5-20 M) (8, 9), only the E1Ca 2 state is primed for transfer of the ␥-phosphoryl group to Asp 351 . As counterions to Ca 2ϩ (3), protons bind to the transport sites in place of Ca 2ϩ , stabilizing the E2 conformation. The fully protonated E2 form (E2H 3 in Scheme 1) is phosphorylated by P i at Asp 351 when the pump works in the reverse mode but cannot be phosphorylated by ATP. Since ATP accelerates P i binding (10) and dephosphorylation (11), modulates P i º HOH exchange (12), and binds fairly tightly to the vanadate complexed E2 form (13), E2H 3 must be able to bind ATP without preventing the access of P i to Asp 351 , suggesting that the ␥-phosphoryl group of the bound ATP is at some distance from Asp 351 in this conformation, in contrast to the E1Ca 2 state. A change in the interaction of bound nucleotide with Asp 351 related to enzyme activation by Ca 2ϩ is clearly demonstrated with TNP-8N 3 -ATP 1 that has

Mutants in which Thr

Residues in conserved motifs 625 The sarcoplasmic reticulum Ca 2ϩ -ATPase 1 from rabbit muscle couples the hydrolysis of ATP to the transport of Ca 2ϩ to the reticular lumen, and the lowering of the sarcoplasmic Ca 2ϩ concentration leads to muscle relaxation. This ion pump is a well studied example of P-type ATPases, the catalytic cycle of which includes a phosphorylated aspartyl intermediate and progression through E1 and E2 states. ATP-dependent phosphorylation takes place upon Ca 2ϩ binding in the E1 form, and hydrolysis of the aspartyl phosphoryl bond occurs in the E2P phosphoenzyme conformation following Ca 2ϩ translocation across the membrane. Two atomic structures of the protein have been elucidated by x-ray crystallography, one in the presence of Ca 2ϩ (designated E1(Ca 2 )) and another in the absence of Ca 2ϩ and presence of the specific inhibitor thapsigargin (E2(TG)) (1, 2). They reveal 10 membrane-spanning helices, connected through a stalk section to the phosphorylation (P) domain in association with nucleotide (N) and actuator (A) domains. The three head domains are loosely attached in E1(Ca 2 ) and closer together, forming a more compact entity, in E2(TG). For ATP-dependent phosphorylation of Asp 351 to occur in the Ca 2ϩ bound E1 state, domain N must close over the phosphorylation site, such that the nucleotide site is brought close to Asp 351 . ATP likely straddles both domains, the nucleoside portion anchored in domain N and the phosphates stretching into domain P (cf. Fig. 1A). Domain P contains the highly conserved segments 625 TGD, 676 FARXXPXXK, and 701 TGDGVND that surround the phosphorylation loop 351 DKTGTLT. Many of these residues have been implicated in ATP binding and catalysis on the basis of chemical labeling and cross-linking experiments, and mutagenesis of Ca 2ϩ -ATPase and related pumps (for review, see Ref.3). Most of them recur at the active site of a large class of soluble phosphohydrolases and phosphotransferases believed to have a similar reaction mechanism (4).