Crystal structures have shown that the conserved TGES loop of the Ca 2؉ -ATPase is isolated in the Ca 2 E1 state but becomes inserted in the catalytic site in E2 states. Here, we have examined the kinetics of the partial reaction steps of the transport cycle and the binding of the phosphoryl analogs BeF, AlF, MgF, and vanadate in mutants with alterations to the TGES residues. The mutations encompassed variation of size, polarity, and charge of the side chains. Differential effects on the Ca 2 E1P 3 E2P, E2P 3 E2, and E2 3 Ca 2 E1 reactions and the binding of the phosphoryl analogs were observed. In the E183D mutant, the E2P 3 E2 dephosphorylation reaction proceeded at a rate as high as one-third that of the wild type, whereas it was very slow in the other Glu 183 mutants, including E183Q, thus demonstrating the need for a negatively charged carboxylate group to catalyze dephosphorylation. By contrast, the Ca 2 E1P 3 E2P transition was accomplished at a reasonable rate with glutamine in place of Glu 183 , but not with aspartate, indicating that the length of the Glu 183 side chain, in addition to its hydrogen bonding potential, is critical for Ca 2 E1P 3 E2P. This transition was also slowed in mutants with alteration to other TGES residues. The data provide functional evidence in support of the proposed role of Glu 183 in activating the water molecule involved in the E2P 3 E2 dephosphorylation and suggest a direct participation of the side chains of the TGES loop in the control and facilitation of the insertion of the loop in the catalytic site. The interactions of the TGES loop furthermore seem to facilitate its disengagement from the catalytic site during the E2 3 Ca 2 E1 transition.The Ca 2ϩ -ATPase of the sarcoplasmic reticulum is a 110-kDa membrane-embedded enzyme that mediates uphill transport of calcium ions from the cytoplasm to the lumen of the sarcoplasmic reticulum at the expense of ATP being hydrolyzed (1, 2). This ATPase, which belongs to the family of P-type ATPases (3), couples ATP hydrolysis with Ca 2ϩ translocation by means of a reaction cycle (Scheme 1) in which phosphorylation and dephosphorylation of the enzyme at a conserved aspartic acid residue alternates with transitions between the phosphorylated or dephosphorylated E1 2 and E2 conformational states (4, 5). The enzyme consists of 10 membrane spanning, mostly helical segments, of which M4, M5, M6, and M8 contribute liganding groups for Ca 2ϩ binding (6 -8), and a large cytoplasmic head piece made up by three distinct domains, named domain P (contains the aspartic acid residue Asp 351 , which becomes phosphorylated during the reaction cycle), domain N (for nucleotide binding), and domain A (for actuator) (6). Crystal structures of the Ca 2ϩ -ATPase with various ligands supposed to stabilize either E1-or E2-like states of the enzyme have indicated that the highly conserved TGES motif of domain A is isolated in E1 and E1P, whereas a rotational movement of domain A brings the loop with the TGES motif into the catalytic site close to the Asp 35...

A cytoplasmic nontransport K؉ /Rb ؉ site in the P-domain of the Na ؉ ,K ؉ -ATPase has been identified by anomalous difference Fourier map analysis of crystals of the [Rb 2 ]⅐E 2 ⅐MgF 4 2؊ form of the enzyme. The functional roles of this third K ؉ /Rb ؉ binding site were studied by site-directed mutagenesis, replacing the side chain of Asp 742 donating oxygen ligand(s) to the site with alanine, glutamate, and lysine. Unlike the wild-type Na ؉ ,K ؉ -ATPase, the mutants display a biphasic K ؉ concentration dependence of E 2 P dephosphorylation, indicating that the cytoplasmic K ؉ site is involved in activation of dephosphorylation. The affinity of the site is lowered significantly (30 -200-fold) by the mutations, the lysine mutation being most disruptive. Moreover, the mutations accelerate the E 2 to E 1 conformational transition, again with the lysine substitution resulting in the largest effect. Hence, occupation of the cytoplasmic K ؉ /Rb ؉ site not only enhances E 2 P dephosphorylation but also stabilizes the E 2 dephosphoenzyme. These characteristics of the previously unrecognized nontransport site make it possible to account for the hitherto poorly understood trans-effects of cytoplasmic K ؉ by the consecutive transport model, without implicating a simultaneous exposure of the transport sites toward the cytoplasmic and extracellular sides of the membrane. The cytoplasmic K ؉ /Rb ؉ site appears to be conserved among Na ؉ ,K ؉ -ATPases and P-type ATPases in general, and its mode of operation may be associated with stabilizing the loop structure at the C-terminal end of the P6 helix of the P-domain, thereby affecting the function of highly conserved catalytic residues and promoting helix-helix interactions between the Pand A-domains in the E 2 state.The essential gradients for Na ϩ and K ϩ across the plasma membranes of mammalian cells are created by the Na ϩ ,K ϩ -ATPase, a membranous enzyme that couples ATP hydrolysis to active extrusion of Na ϩ from the cells and uptake of K ϩ at a stoichiometry of three Na ϩ ions exchanged for two K ϩ ions per ATP molecule utilized (1, 2). The Na ϩ ,K ϩ -ATPase belongs to the family of P-type ATPases characterized by the formation of a phosphorylated enzyme intermediate through transfer of the ␥-phosphate of ATP to a conserved aspartate residue in the enzyme. The ATP hydrolysis is tightly coupled to ion translocation, because Na ϩ binding from the cytoplasmic side triggers phosphorylation of the enzyme from ATP, whereas K ϩ binding from the extracellular side leads to rapid dephosphorylation (3). Recently, the structure of the Na ϩ ,K ϩ -ATPase was determined at 3.5 Å resolution (4), revealing two Rb ϩ ions bound as K ϩ congeners close together in a pocket formed by the transmembrane segments M4, 2 M5, M6, and M8 of the catalytic ␣-subunit. The ␣-subunit consists of 10 transmembrane segments, M1-M10, and a large cytoplasmic part containing three distinct domains denoted A (actuator), N (nucleotide-binding), and P (phosphorylation), by analogy to the closely related sarco(end...

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 3 E 2 P and E 2 P 3 E 2 reactions) and Ca 2؉ binding (E 2 3 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 3 E 2 P transition were small, suggesting that the nucleotide that accelerates Ca 2 E 1 P 3 E 2 P binds differently from that modulating the E 2 P 3 E 2 and E 2 3 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 3 E 2 and acceleration of the latter reaction, indicating that ATP normally modulates E 2 P 3 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 .The sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (1) is a membrane-bound energy transducer ("nanomotor") that couples ATP hydrolysis with Ca 2ϩ 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 Ca 2 E 1 /Ca 2 E 1 P and E 2 /E 2 P forms (2, 3). In recent years, several high resolution crystal structures of the Ca 2ϩ -ATPase, each thought to represent a particular intermediate state in the pump cycle, have been determined (4 -11). The Ca 2ϩ -ATPase consists of a membrane-spanning domain of 10 mostly helical segments and a large cytoplasmic headpiece, comprising three distinct domains, named N (nucleotide binding), P (phosphorylation), and A (actuator) (4). In the Ca 2 E 1 and Ca 2 E 1 P conformations, the catalytic ATPbinding site is made up by residues in the N-and P-domains, and during the Ca 2 E 1 P 3 E 2 P transition the departing ADP molecule is replaced by the TGES loop of the A-domain, which subsequently assists in catalysis of E 2 P dephosphorylation (8,9,12). In addition to being the phosphorylating substrate in the E 1 state, ATP exerts modulatory effects on various steps of the Ca 2ϩ -ATPase cycle (boxed ATP in Scheme 1). Hence, the Ca 2 E 1 P 3 E 2 P, E 2 P 3 E 2 , and E 2 3 Ca 2 E 1 transitions are all accelerated by the binding of ATP or MgATP in a nonphosphorylating mode (13-23). The apparent affinity for the nucleotide is generally lower when it binds in the nonphosphorylating modulatory mode as compared with the phosphorylating mode, but it varies depending on the step being modulated. 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 s...

Mutants with alteration toThe sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (1) is an energytransducing enzyme of the P-type that couples hydrolysis of ATP to translocation of Ca 2ϩ from the cytosol to the endoplasmic reticulum. In this control of cytosolic Ca 2ϩ concentration, the Ca 2ϩ -ATPase plays a vital role in cellular activation events, such as muscle contraction, hormone secretion, immune responses, cell migration, and protein synthesis. Ca 2ϩ transport is coupled to ATP hydrolysis by a reaction cycle (Scheme 1), in which the enzyme is transiently phosphorylated at a conserved aspartic acid residue and undergoes major conformational changes (2, 3).In recent years, several high resolution crystal structures of the Ca 2ϩ -ATPase, each thought to represent a particular intermediate state in the pump cycle, have been solved (4 -9). The Ca 2ϩ -ATPase consists of a membrane-spanning domain of ten helical segments and a large cytoplasmic head piece, comprising three distinct domains, named "N" (nucleotide binding), "P" (phosphorylation), and "A" (actuator). By combining crystallographic data with functional changes in site-specific mutants, an increasingly detailed picture of the mechanisms of energy interconversion and ion translocation in the Ca 2ϩ -ATPase is emerging. Thus, the catalytic function in E1 (autokinase activity) and E2 forms (autophosphatase activity), the movement of Ca 2ϩ ions across the membrane, as well as the major rate-limiting conformational changes of the cycle, i.e. E2 3 E1 and E1P 3 E2P, can all be understood on the basis of the sequential gathering and displacement of certain conserved amino acid motifs in domains N and A relative to the catalytic site in domain P and the coupling of these events to rearrangements of the transmembrane helices containing the high affinity Ca 2ϩ sites. In the present study, we address the role of Asn 706 at the catalytic site of Ca 2ϩ -ATPase and revisit a previously examined mutant, E183A (10). Asn 706 and Glu 183 reside in the conserved 701 TGDGVND 707 (domain P) and 181 TGES 184 (domain A) motifs, respectively, and both residues are found in all known P-type ATPases (11). In fact, Asn 706 is highly conserved even in the superfamily of phosphohydrolases and phosphotransferases (the HAD superfamily), which, given the similarities in reaction mechanism, protein sequence, and structural architecture of the catalytic site, are believed to share a common evolutionary ancestor with the phosphorylation domain of the P-type ATPases (12-14). The side chains of Asn 706 and Glu 183 are both centrally located at the catalytic site in the E2 forms of Ca 2ϩ -ATPase, close to the phosphorylated aspartate, Asp 351 (6, 7, 9). In E1 conformations, Glu 183 has departed the phosphorylation site, whereas Asn 706 retains its close proximity to Asp 351 (4,5,7,8). In our previous study of Glu 183 (10), we demonstrated that substitution of the glutamate with alanine leads to a much reduced rate of both E2P hydrolysis and of the reverse phosphorylation of E2 with P i , suggesti...