Participation of hydrogen ion in the calcium-induced conformational transition of 4-nitro-2,1,3-benzoxadiazole-labeled sarcoplasmic reticulum ATPase

Wakabayashi, Shigeo; Ogurusu, Tarou; Shigekawa, Munekazu

doi:10.1021/bi00499a006

Cited by 33 publications

(32 citation statements)

References 31 publications

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“…However, from a kinetic point of view the rate of the E2 to Ca 2 E1 transition is generally thought to be rate-limited not by Ca 2ϩ binding per se but by a conformational transition occurring either after binding of the first Ca 2ϩ ion (78), or, alternatively before binding of Ca 2ϩ , an event accompanied by deocclusion of intramembraneous protons bound to the translocation sites in the E2 state (80,81). Thus, the slowing down of the E2 to Ca 2 E1 transition in ADA ATPase seems to favor an indirect effect of the mutation, as further discussed below.…”

Section: Discussionmentioning

confidence: 99%

Involvement of the L6–7 Loop in SERCA1a Ca2+-ATPase Activation by Ca2+ (or Sr2+) and ATP

Lenoir

Picard

Møller

et al. 2004

Journal of Biological Chemistry

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Wild-type (WT) and the double mutant D813A,D818A (ADA) of the L6 -7 loop of SERCA1a were expressed in yeast, purified, and reconstituted into lipids. This allowed us to functionally study these ATPases by both kinetic and spectroscopic means, and to solve previous discrepancies in the published literature about both experimental facts and interpretation concerning the role of this loop in P-type ATPases. We show that in a solubilized state, the ADA mutant experiences a dramatic decrease of its calcium-dependent ATPase activity. On the contrary, reconstituted in a lipid environment, it displays an almost unaltered maximal calcium-dependent ATPase activity at high (millimolar) ATP, with an apparent affinity for Ca 2؉ altered only moderately (3-fold). In the absence of ATP, the true affinity of ADA for Ca 2؉ is, however, more significantly reduced (20 -30-fold) compared with WT, as judged from intrinsic (Trp) or extrinsic (fluorescence isothiocyanate) fluorescence experiments. At low ATP, transient kinetics experiments reveal an overshoot in the ADA phosphorylation level primarily arising from the slowing down of the transition between the nonphosphorylated "E2" and "Ca 2 E1" forms of ADA. At high ATP, this slowing down is only partially compensated for, as ADA turnover remains more sensitive to orthovanadate than WT turnover. ADA ATPase also proved to have a reduced affinity for ATP in studies performed under equilibrium conditions in the absence of Ca 2؉ , highlighting the long range interactions between L6 -7 and the nucleotide-binding site. We propose that these mutations in L6 -7 could affect protonation-dependent winding and unwinding events in the nearby M6 transmembrane segment.Muscle relaxation occurs by of the removal of calcium from the cytosol and its accumulation into sarcoplasmic reticulum (SR) 1 through an active transport that consumes ATP (1, 2). In fast twitch muscle this transport is achieved by the SERCA1a isoform of Ca 2ϩ -ATPase (3), with a stoichiometry of two calcium ions transported per ATP molecule hydrolyzed (see reviews in Refs. 4 and 5). SR Ca 2ϩ -ATPase belongs to the family of P-type ATPases, which transport cations such as H ϩ , Na ϩ , K ϩ , or Ca 2ϩ across the membrane and share a common mechanism that involves autophosphorylation of the protein (see review in Ref. 6): transport is achieved through a reversible cycle, during which the ATPase is thought to sequentially adopt two main conformations (7, 8), E1 and E2, with a high and low affinity for the transported ion, and the overall cycle corresponds to completion of the following scheme: E2 3 Ca 2 E1 3 Ca 2 E1 ϳ P 3 E2P 3 E2 (for review, see Refs. 9 and 10). Much effort has gone into determining the structural organization of SR Ca 2ϩ -ATPase from primary structure predictions (11), electron microscopy (12), and three-dimensional crystallogenesis (13-15) (for a recent review see Ref. 16). Finally, the structure has been solved at the atomic level, first in a Ca 2 E1 state (14) and then in a thapsigargin-stabilized E2 state (15). T...

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Section: Discussionmentioning

confidence: 99%

Involvement of the L6–7 Loop in SERCA1a Ca2+-ATPase Activation by Ca2+ (or Sr2+) and ATP

Lenoir

Picard

Møller

et al. 2004

Journal of Biological Chemistry

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“…7). Because other partial reaction steps that contribute to rate limitation of the overall reaction, such as the E 1 ϳP(Ca 2 ) to E 2 P and E 2 to E 1 Ca 2 transitions, tend to be accelerated at alkaline pH (49,52,59), and the dephosphorylation of E 2 P remained relatively fast in the SERCA3 enzymes under these conditions, a higher turnover rate could be reached at alkaline pH in the SERCA3 enzymes as compared with SERCA1a. Furthermore, because the rate of E 2 P dephosphorylation was higher in SERCA3b than in the other SERCA3 enzymes at alkaline pH, SERCA3b reached the highest turnover rate at pH optimum (Figs.…”

Section: Fig 13 Vanadate Inhibition During Steady-state Atp Hydrolymentioning

confidence: 99%

Dissection of the Functional Differences between Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA) 1 and 3 Isoforms by Steady-state and Transient Kinetic Analyses

Dode

Vilsen

Baelen

et al. 2002

Journal of Biological Chemistry

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Steady-state and transient-kinetic studies were conducted to characterize the overall and partial reactions of the Ca 2؉ -transport cycle mediated by the human sarco(endo)plasmic reticulum Ca 2؉ -ATPase 3 (SERCA3) isoforms: SERCA3a, SERCA3b, and SERCA3c. Relative to SERCA1a, all three human SERCA3 enzymes displayed a reduced apparent affinity for cytosolic Ca 2؉ in activation of the overall reaction due to a decreased E 2 to E 1 Ca 2 transition rate and an increased rate of Ca 2؉ dissociation from E 1 Ca 2 . At neutral pH, the ATPase activity of the SERCA3 enzymes was not significantly enhanced upon permeabilization of the microsomal vesicles with calcium ionophore, indicating a difference from SERCA1a with respect to regulation of the lumenal Ca 2؉ level (either an enhanced efflux of lumenal Ca 2؉ through the pump in E 2 form or insensitivity to inhibition by lumenal Ca 2؉ ). Other differences from SERCA1a with respect to the overall ATPase reaction were an alkaline shift of the pH optimum, increased catalytic turnover rate at pH optimum (highest for SERCA3b, the isoform with the longest C terminus), and an increased sensitivity to inhibition by vanadate that disappeared under equilibrium conditions in the absence of Ca 2؉ and ATP. The transient-kinetic analysis traced several of the differences from SERCA1a to an enhancement of the rate of dephosphorylation of the E 2 P phosphoenzyme intermediate, which was most pronounced at alkaline pH and increased with the length of the alternatively spliced C terminus.Sarco(endo)plasmic reticulum Ca 2ϩ -ATPases (SERCAs) 1 are single-subunit membrane-spanning P-type ATPases that mediate the uphill transport of cytoplasmic Ca 2ϩ into the lumen of intracellular stores with a stoichiometry of two Ca 2ϩ per ATP hydrolyzed (1-3). Studies of the SERCA1a enzyme have shown that Ca 2ϩ transport and ATP utilization are coupled through long-range intramolecular interactions between the 10-helix transmembrane domain containing the Ca 2ϩ binding sites and the cytoplasmic actuator, phosphorylation, and nucleotidebinding domains (1-4). In the reversible catalytic cycle (5), the binding of the two calcium ions with high affinity from the cytoplasmic side of the membrane triggers the phosphorylation of Asp 351 by ATP, whereas the dephosphorylation occurs when the translocated calcium ions have been released from lowaffinity lumenally facing sites in exchange with protons being countertransported (Scheme 1).The SERCAs have long been known as key enzymes in the cytoplasmic Ca 2ϩ signaling events, and more recent studies have also drawn attention to the importance of the luminal Ca 2ϩ content for Ca 2ϩ signaling, folding and processing of newly synthesized proteins, and control of cell growth and apoptosis (6 -9). In addition to the functionally well characterized SERCA1a isoform, the 3 human SERCA genes encode at least 8 other SERCA proteins by alternative splicing (10 -15). Whereas SERCA2b seems to be a ubiquitously expressed housekeeping enzyme, the other isoforms are limited to specific c...

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“…It has been shown that Ca 2+ binding occurs in exchange with H + [13,14], and the stoichiometry of exchange is dependent on medium pH [15]. Ca 2+ /H + exchange is of interest both for the involvement of carboxylic amino acid chains in Ca 2+ binding at the start of the ATPase cycle, and for the subsequent release of bound Ca 2+ following ATP utilization and enzyme turnover [16,17].…”

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confidence: 99%

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

Zafar¹,

Hussain²,

Liu³

et al. 2008

Archives of Biochemistry and Biophysics

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Ligand binding to transport sites constitutes the initial step in the catalytic cycle of transport ATPases. Here, we consider the well characterized Ca 2+ ATPase of sarcoplasmic reticulum (SERCA) and describe a series of Ca 2+ binding isotherms obtained by equilibrium measurements in the presence of various H + and Mg 2+ concentrations. We subject the isotherms to statistical mechanics analysis, using a model based on a minimal number of mechanistic steps. The analysis allows satisfactory fits and yields information on occupancy of the specific Ca 2+ sites under various conditions. It also provides a fundamental method for analysis of binding specificity to transport sites under equilibrium conditions that lead to tightly coupled catalytic activation. KeywordsCa 2+ ATPase; Ca 2+ binding; H + /Ca 2+ exchange; Statistical analysis Ligand binding to transport sites constitutes the initial step in the catalytic cycle of transport ATPases. The characteristics of this binding are of utmost importance, as they determine catalytic activation, specificity and stoichiometry of transport and countertransport, and efficiency of ATP utilization. They also correspond to structural features of the enzyme protein with regard to binding affinity and specificity, as well as conformational consequences of binding resulting in catalytic activation. We provide here a detailed analysis of Ca 2+ binding to the Ca 2+ transport ATPase of sarco-endoplasmic reticulum (SERCA) [1,2]. The SERCA isoform of skeletal muscle is a well characterized enzyme [3,4] that utilizes the free energy of ATP for Ca 2+ transport against a concentration gradient. The functional unit is a protein monomer comprising 994 amino acid residues. The sequence is folded into a cluster of 10 segments forming a transmembrane region, and three relatively large domains ("N", "P" and "A") protruding from the cytosolic surface of the membrane [5,6]. The ATPase cycle begins with high affinity binding of Ca 2+ derived from the cytosolic medium ("outside"), followed by ATP utilization to form a phosphorylated enzyme intermediate. Isomerization of the phosphoenzyme intermediate is then coupled to active transport of the bound Ca 2+ across the membrane ("inside"). Hydrolytic cleavage of the phosphoenzyme is the final step that allows enzyme turnover. The high Ca 2+ affinity state of the enzyme is generally referred to as E 1 , and the low affinity as E 2 . Methods Ca 2+ binding and ATPase measurementsVesicular fragments of sarcoplasmic reticulum membrane were obtained from rabbit hind leg muscle as previously described [19]. SERCA accounts for 50-60% of total protein in this preparation. Total protein was determined by the Folin method, standardized with serum albumin.Calcium binding was measured under equilibrium conditions by molecular sieve chromatography [7,20] We now discuss the energies associated with the proposed SERCA species. The energy of an unoccupied species (E) is set as the reference and the energies of all other species are measured with respect to it. ...

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Participation of hydrogen ion in the calcium-induced conformational transition of 4-nitro-2,1,3-benzoxadiazole-labeled sarcoplasmic reticulum ATPase

Cited by 33 publications

References 31 publications

Involvement of the L6–7 Loop in SERCA1a Ca2+-ATPase Activation by Ca2+ (or Sr2+) and ATP

Involvement of the L6–7 Loop in SERCA1a Ca2+-ATPase Activation by Ca2+ (or Sr2+) and ATP

Dissection of the Functional Differences between Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA) 1 and 3 Isoforms by Steady-state and Transient Kinetic Analyses

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

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