Substantia. An International Journal of the History of Chemistry 3(1): 9-17, 2019 Firenze University Press www.fupress.com/substantia ISSN 1827-9635 (print) | ISSN 1827-9643 (online) | DOI: 10.13128/Substantia-68 Citation: G. Inesi (2019) Similarities and contrasts in the structure and func- tion of the calcium transporter ATP2A1 and the copper transporter ATP7B. Substantia 3(1): 9-17. doi: 10.13128/ Substantia-68 Copyright: © 2019 G. Inesi. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/substantia) and distribuited under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Research Article Similarities and contrasts in the structure and function of the calcium transporter ATP2A1 and the copper transporter ATP7B Giuseppe Inesi California Pacific Medical Center Research Institute, 32 Southridge Rd West, Tiburon CA 94920, USA E-mail: giuseppeinesi@gmail.com Abstract. Ca2+ and Cu2+ ATPases are enzyme proteins that utilize ATP for active trans- port of Ca2+ or Cu2+ across intracellular or cellular membranes.1-4 These enzymes are referred to as P-type ATPases since they utilize ATP through formation of a phospho- rylated intermediate (E-P) whose phosphorylation potential affects orientation and affinity of bound cations by means of extended conformational changes. Thereby spe- cific cations are transported across membranes, forming transmembrane gradients in the case of Ca2+, or accepting Cu2+ from delivering proteins on one side of the mem- brane and releasing it to carrier proteins on the other side. Binding of Ca2+ or Cu2+ is required for enzyme activation and utilization of ATP by transfer of ATP terminal phosphate to a conserved aspartate residue. The ATPase protein is composed of a transmembrane region composed of helical segments and including the cation binding site (TMBS), and a cytosolic headpiece with three domains (A, N and P) containing the catalytic and phosphorylation site. The number of helical segments and the cytosol- ic headpieces present significant differences in the two enzymes. In addition, details of transmembrane cation extrusion are different. The Ca2+ and Cu2+ ATPase sustain vital physiological functions, such as muscle contraction and relaxation, activation of several cellular enzymes, and elimination of excess cation concentrations. A historic review of studies on chemical and physiological mechanisms of the Ca2+ and Cu2+ ATPase is pre- sented. Keywords. Calcium ATPase, Copper ATPase, Cation Active Transport. THE CALCIUM TRANSPORT ATPASE The Ca2+ATPase (SERCA) is a mammalian membrane bound protein sustaining Ca2+ transport and involved in cell Ca2+ signaling and homeo- stasis. It is made of a single polypeptide chain of 994 amino acid residues distributed in ten trans-membrane segments (M1 – M10) and a cytosolic headpiece including three distinct domains (A, N and P) that are directly involved in catalytic activity (Fig 1).5 The N domain contains residues such (Phe-487) interacting with the adeno- sine moiety of ATP whereby the ATP substrate is cross-linked to the P domain. 10 Giuseppe Inesi The P domain contains a residues (Asp-351) undergoing phosphorylation to yield a phosphorylated intermediate (E-P), a residue (Asp-703) coordinating Mg2+, and other features characteristic of P-type ATPases. The A domain contains the signature sequence 181TGE that provides cata- lytic assistance for final hydrolytic cleavage of (E-P). Coop- erative and sequential binding of Ca2+ involved in catalytic activation and transport (Figs. 1 and 3) occurs on sites I and II located within the trans-membrane region.6,7 Ca2+ATPases (SERCA1 and SERCA2) are associ- ated with intracellular membranes of skeletal and car- diac muscle (sarcoplasmic reticulum: SR), and especially high concentrations with the skeletal muscle SR. There- fore, isolation of vesicular fragments of skeletal SR yields concentrated and fairly pure protein, shown by very fre- quent particles corresponding to ATPase protein visual- ized by electron microscopy (Fig 2 left panel), and prom- inent ATPase component visualized by electrophoresis (Fig 2, right panel). This preparation is very convenient for functional and structural characterization of the ATPase.8 In fact, it was demonstrated (8) with this preparation that, at equi- librium and in the absence of ATP, SERCA binds two Ca2+ per mole, with high cooperativity and high affinity (2.3 x 106 M-1) (Fig. 3a) at neutral pH, although the affin- ity is lower at low pH and higher at higher pH.9 When ATP is added to SR vesicles pre-incubated with Ca2+ in rapid kinetic experiments (Fig 3b), the bound Ca2+ fac- ing the outer medium disappears soon (becomes non available to isotopic exchange, i.e., occluded), indicating that the outer opening of the binding cavity closes to the outside medium as soon as a first reaction product with ATP is formed. Pi release and further Ca2+ uptake then occur fol- lowing a delay, indicating that trans-membrane Ca2+ release and hydrolytic cleavage of EP occur after a slow step and, soon after that, further cycles contribute to steady state activity.10 Based on these kinetic observations, a diagram is shown in Fig. 4, where the basal enzyme is indicated as 2H+.E2. Following 2Ca2+ binding in exchange for 2H+, the active enzyme is referred to as E1.2Ca+. Following binding and utilization of ATP, the resulting phospho- enzyme is indicated as ADP.E1-P.2Ca2+. Upon release of ADP, the free energy associated with this intermediate is utilized for a slow conformational change yielding trans- membrane release of bound Ca2+ in exchange for 2 H+, Figure 1. Aminoacid sequence and two dimensional folding model of the SERCA1 Ca2+ ATPase.5 See text for explanations. Figure 2. Purified vesicular fragments of sarcoplasmic reticulum membrane shown by negative staining on electron microscopy. On the right side, electrophoretic analysis demonstrates that the protein composition consists almost entirely of Ca2+ ATPase.8 Figure 3a. Ca2+ binding to SR ATPase under equilibrium con- ditions, in the absence of ATP. The stoichiometry of binding is 2 Ca2+ per ATPase, with a binding constant of 2.3 x 106 M-1, and high cooperativity.9 Figure 3b. Pre-steady state activity of SR vesi- cles started by addition of ATP in the presence of Ca2+. Note that upon addition of ATP a rapid burst of EP formation occurs and, at the same time, 2 Ca2+ per ATPase become occluded. Steady state Pi production and further Ca2+ uptake then follow, with a ratio of 2 Ca2+ per Pi produced.10 11Similarities and contrasts in the structure and function of the calcium transporter ATP2A1 and the copper transporter ATP7B followed by hydrolytic cleavage of Pi and return to the basic 2H+.E2 state. The reaction scheme in Fig. 4 outlines a specific exchange of 2 Ca2+ for 2H+ in the 2H+.E2 state and 2H+ for 2 Ca2+ in the E2-P.2Ca+ state. Clear evidence for this exchange was obtained with SERCA reconstituted in phospholipids vesicles that do not allow trans-membrane passive leak of charge which occurs in native SR membranes (except for transported Ca2+). Ca2+ and H+ concentrations and electrical poten- tial were then measured with appropriate sensors (Fig. 5). It was found that addition of ATP was accompanied by Ca2+ uptake and stoichiometric H+ extrusion, as well as formation of electrical potential.11 The important role of H+ at the Ca2+ sites was also demonstrated in experiments with native mem- brane vesicles, as it was found that phosphorylation of ATPase with Pi can be obtained only at acid pH. This indicates that upon 2 H+ binding to E2 (in exchange for Ca2+ if present) the resulting 2H+.E2 acquires a specific conformation and free energy to allow phosphorylation with Pi, i.e. reversal of the E2-P.2Ca2+ to 2H+.E2 step in the ATPase reaction cycle.12 Pioneering and highly informative crystallography by Toyoshima et al. revealed detailed structural infor- mation on the molecular structure of the entire mol- ecule.13 Nucleotide and phosphorylation domains of the Ca2+ ATPase, relative to different stages of the enzyme cycle, are represented in Fig 6.14 In the figure, the struc- ture and conformational states of the Ca2+ ATPase in the presence and absence of Ca2+, substrate and prod- uct analogs are represented, with reference to E1. 2Ca2+, E1.AMPPCP, E-2.AlF4.(TG), and E2.(TG).ATP, where TG (thapsigargin, a highly specific and potent SERCA inhibitor) is used to stabilize E2.13, 15, 16, 17 Color changes gradually from the N-terminus (blue) to the C-terminus (red). The two Ca2+ (I and II) bound to the high affinity transmembrane site are circled when present. The two bound Ca2+ undergo vectorial release in E2.AlF4.(TG), as the binding sites undergo a change in affinity and orientation. Three key residues (E183 in the A domain, D351 and D703 in the P domain) are shown in ball-and- stick. Note the positional change of headpiece domains in the various conformations. Note the nucleotide bind- ing to the N domain, and variable relationship of the nucleotide phosphate chain (and Mg2+) with the P and A domains. As described above, kinetic and structural informa- tion yields a detailed understanding of the Ca2+ ATPase catalytic and transport cycle as outlined in Fig. 4. Figure 4. Diagram outlining the sequential reactions of a Ca2+ ATPase cycle at neutral pH. The cycle starts with the enzyme in basal conformation, with H+ bound at the specific Ca2+ exchange site (2H.E2). Upon H+ dissociation, 2Ca2+ bind and the enzyme is activated (E1.2Ca). ATP then leads to formation of the high poten- tial phosphorylated intermediate (ADP.E1*P.2Ca). Following dis- sociation of ADP, the phosphorylated intermediate uses its poten- tial for a conformational change reducing affinity and orientation of bound calcium. 2 Ca2+ are then dissociation in exchange for 2 H+. The residual phosphoenzyme then undergoes hydrolytic cleav- age with release of Pi, and returns to the basal conformation with H+ bound (2H.E2). The stoichiometry of H+ binding is 2 per E at neutral pH. At high pH, less or no H+ exchanges for Ca2+. Thereby Ca2+ is not released before Pi cleavage, and the enzyme undergoes an uncoupled cycle. Figure 5. ATP dependent Ca 2+ uptake, H+ countertransport and development of transmembrane electrical potential in reconsti- tuted SERCA proteoliposomes. The proteoliposomes were placed in a neutral pH medium, containing 100 mM K2SO4, 50 microM CaCl2, and color reagents for detection of Ca2+, pH and electro- chemical gradients. The reaction was started by the addition of 0.2 mM ATP, and followed by differential absorption spectrometry.11 12 Giuseppe Inesi THE COPPER TRANSPORT ATPASE Bacterial and mammalian copper ATPases sustain active transport of copper by utilization of ATP . The mammalian Cu+ ATPases include isoforms (ATP7A and ATP7B) that are involved in copper transfer from enterocytes to blood, copper export from the liver to the secretory pathways for incorporation into metal- loproteins, and general copper homeostasis.18,19 Genet- ic defects of ATP7A and ATP7B are related to human Menkes and Wilson diseases.20, 21, 22 Cu2+ ATPases present functional analogies to the Ca2+ ATPases, but specific differences as well. A com- parison of SERCA and ATP7B bidimentional folding models (Fig. 7) shows that ATP7B comprises eight (rath- er than ten) transmembrane segments that include the copper binding site (TMBS) for catalytic activation and transport, and a headpiece comprising the N, P and A domains with conserved catalytic motifs analogous to SERCA. A specific feature of ATP7B (less prominent in ATP7A, and absent in the bacterial copper ATPase) is an amino-terminal extension (NMBD) with six cop- per binding sites in addition to those in the TMBD. An additional feature is the presence of serine residues (Ser- 478, Ser-481, Ser1211, Ser-1453 in ATB7B) undergoing kinase assisted phosphorylation.23 The native abundance of copper ATPase is quite low and, in order to accomplish biochemical experimenta- tion, larger quantities were obtained by heterologous expression in insect or mammalian cells.24, 25 It was found that addition of ATP to microsomes expressing heterologous ATP7B yields two fractions of phosphoryl- Figure 6. Sequence of conformational states of the calcium ATPase in the presence (E1.2Ca2+), following nucleotide (analog) substrate binding (E1.AMPPCP), following enzyme phosphorylation (AlF4 analog) and Ca2+ release (E2.AlF4.TG) and in absence of Ca2+ with bound ATP (E2(TG).ATP).14 Figure 7. Two-dimensional folding models of the Ca2+ ATPase (SERCA1) and Cu+ ATPase (ATP7B) sequence. The diagram shows ten SERCA or eight ATP7B transmembrane domains including the calcium or copper binding sites (TMBS) involved in enzyme activa- tion and cation transport. The extra-membranous regions of both enzymes comprises a nucleotide binding domain (N), the P domain with several conserved residues (in yellow) including the aspartate (Asp351 and Asp1027) undergoing phopsphorylation to form the catalytic intermediate (EP), and the A domain with the TGE con- served sequence involved in catalytic assistance of EP hydrolytic cleavage. The His1069 residue whose mutation is frequently found in the Wilson disease is shown in the ATP7B N domain. Specific features of ATP7B are the N-metal Binding Domain (NMBD) extension with six copper binding sites, and serine residues under- going Protein Kinase assisted phosphorylation (Ser478, Ser 481, Ser1211, Ser1453).23 13Similarities and contrasts in the structure and function of the calcium transporter ATP2A1 and the copper transporter ATP7B ated ATPase protein, one acid labile corresponding to phosphoenzyme intermediate, and the other acid stable and dependent on kinase assisted phosphorylation. Acid labile phosphoenzyme is faster, and is not observed fol- lowing mutation of the conserved aspartate (S1024) at the catalytic site, or following mutation of the trans- membrane copper binding site (TMBD). Kinase assisted formation of alkali resistant phosphorylation is slower, involves Ser478, S481, Ser1121 and Ser1453, and is not observed in the presence of protein kinase inhibitors. Interestingly, it is not observed following mutation of the trans-membrane copper binding site (TMBD), indicating a dependence on enzyme activation (E2 to E1) transition. Specific features of copper ATPase following addi- tion of ATP are shown in Fig 8, to demonstrate the dif- ference in phosphorylation of aspartate and serines in the copper ATPase. The time course of ATP7B follow- ing addition of ATP is shown in Fig 8A, with total phos- phoenzyme (black squares) including acid and alkaline resistant (dark squares, including aspartate phosphoen- zyme intermediate and phospho-serines), acid resistant (dark circles, i.e. aspartate phosphoenzyme intermedi- ate) and alkaline resistant (light squares, i.e. phosphor- serines). It is shown in Fig 8B that no alkaline resistant phospoenzyme (i.e. phospho-serines, light squares) is observed if protein kinase inhibitor is present, and in Fig 8C no acid resistant aspartate phosphoenzyme (dark cir- cles) is observed when D1027N ATP7B is used. By com- parison, it is shown in Fig 8D that WT SERCA under- goes only acid stable aspartate phosphorylation, and no alkali resistant serine (light squares) phosphorylation, i.e. the acid stable accounts for total phosphorylation .25 An estimate of Cu2+ transport following phosho- rylation of ATP7B with ATP was obtained by comparing microsomes of COS-1 expressing Ca2+ ATPase (SERCA) or Cu+ ATPae (ATP7B) absorbed on a solid supported membrane (SSM). The SSM consists of an alkanethiol monolayer covalently bound to a gold electrode via the sulfur atom and a phospholipids monolayer on top of it.26, 27, 28 The adsorbed protein is activated by addition of ATP in the presence of a medium supporting ATPase activity. Related electrogenic events are recorded as cur- rent transients due to flow of electrons along the exter- nal circuit toward the electrode surface, as required to compensate for the potential difference across the vesicular membrane produced by displacement of posi- tive charge upon vectorial translocation in the direc- tion of the SSM electrode. When ATP is added to the membrane bound ATPase absorbed on the SSM in the presence of Ca2+ or Cu2+, a current transient is obtained due to vectorial translocation of bound Ca2+ or Cu2+ in the direction of the SSM electrode after phoshoenzyme formation by utilization of ATP. In these experimen- tal conditions, the electrogenic signal generated within the first enzyme cycle is observed.29 It is shown in Fig 9A that in experiments with SERCA that the charge transfer observed at neutral pH is much reduced at acid pH. On the other hand, the charge transfer observed with ATP7B is significantly slower, and is not changed by alkaline or acid pH (Fig 9B). This difference is due to the lack of Cu2+/H+ exchange in the cation bind- ing and release sites of the copper ATPase, as opposed to the requirement of Ca2+/H+ exchange in the calcium ATPase. A crystallographic view of the copper ATPase pro- tein and of the copper transport pathway across the membrane was obtained through LpCopA crystalliza- tion, trapped in the E2.Pi, as compared with E2P state.31 The two states show the same conduit, appearing equiva- lent and open to the extracellular side, in contrast to the Figure 8. Phosphorylation of WT ATP7B (A, B), ATP7B D1027N mutant (C), and WT SERCA (D), in the absence (A, C and B) and in the presence of Proteinase K inhibitor. Microsomes obtained from COS-1 cells sustaining expression of the various ATPases were incubated with 50 microM (gamma -32P)ATP in a reaction mixture sustaining enzyme activiy at 30 oC, in the absence (A, C and D) or in the presence (B) of PKD inhibitor. Electrophoresis was then performed at acid pH to measure total phosphoproteins (black squares), or alkali pH to eliminate alkali labile phosphoenzyme and assess alkali resistant serine phosphorylation (empty squares). The difference (given in in the absence (A, C and D) or in the presence (B) of PKD inhibitor. Electrophoresis was then performed at acid pH to measure total phosphoproteins (black squares), or alkali pH to eliminate alkali labile phosphoenzyme and assess alkali resist- ant serine phosphorylation (solid black circles) corresponds to the phosphorylated aspartate enzyme intermediate.25 14 Giuseppe Inesi calcium ATPase where the E2.Pi state is occluded. In Fig 10a the A, P and N domains are colored in yellow, blue and red, respectively. The black arrows mark the copper transport pathway. In Fig 10b the E2P (pink) and E2.Pi (green) states are compared, showing movements of the extracellular domains (arrows), while the transmem- brane domain remains rigid in two states, in contrast to the calcium ATPase where the E2.Pi becomes occluded. Fig 10c shows a close up of the extrusion pathway with the opening from the copper high affinity coordinating residues Cys382, Cys384, and Met717 shown as a red surface, with crystallographic water molecule shown as red spheres. A diagrammatic comparison of the calcium and copper ATPases is shown in Fig 11, where the sequential conformational transitions of the catalytic and transport cycle are compared for calcium and copper ATPases.30 We then see that the two calcium ions exit the ATPase from the E2P state, and the ion exit pathway closes con- comitantly to hydrolytic cleavage of Pi and transition to the E2.Pi state. On the other hand, the copper ions exit the ATPase from the E2P state, but the exit pathway remains open in the E2.Pi state, and closes only in the E2 state after release of Pi. Considering experimental results and modeling shown above, there seems to be a clear parallel between the difference in cation/proton exchange, and the con- formational outcome in the exit pathways following cation release in the two ATPases. It is apparent that the closure of the release pathway in the calcium ATPase is due to H+ binding in exchange for Ca2+, and a conse- quent conformational effect on the E2.Pi state. The path- way closure in the copper ATPase occurs only following release of Pi and acquisition of the E2 conformation. A further distinctive feature of the copper ATPase is the effect of phosphorylation of serine residues catalyzed by Proteine Kinase D.25 In experiments with micro- somes of COS1 cells or hepatocytes expressing ATP7B it was found that utilization of ATP by ATP7B includes autophosphorylation of an aspartyl residue serving as the specific catalytic intermediate, as well as phospho- rylation of serine residues catalyzed by Protein Kinase D. It is shown in Fig 12 A that ATP7B (stained in green) interacts first with TransGolgi network (blue) in perinu- clear (nuclei red) location and, in the presence of Cu2+, is transferred to intracellular trafficking vesicles. It is shown in Fig12 B that the trafficking is not interfered with by mutation of the TMBD Asp1027 (whose phos- phorylation serves as phosphoenzyme intermediate). On the other hand, trafficking is interfered by Ser478, 481, 1121 and 1453 mutations in the NMD (Fig12C), by TMBS copper site mutation (Fig 12D), and by muta- tion of the 6th NMBD copper site mutation (Fig 12 E). This demonstrates that the NMD, absent in the calcium ATPase, plays a determinant role in conformational adaptations required for functions of the copper ATPase. Figure 9. Charge measurements measured with enzymes absorbed on solid supports member (SSM). A: Current transients follow- ing addition of ATP to SERCA in a reaction mixture including 10 microM free Ca2+ and 100 mM KCl at pH 7.0 (solid line) or pH 7.8 (dotted lines). C: Current transients after addition to ATP on ATP7B in a reaction mixture containing 5 microM Cu+ and 100 mM KCl at pH 6.0 (solid line) or 7.8 (dotted line).28 Figure 10. Diagrammatic representation showing that crystal waters of the E2-BeF3- structure support the copper release path- way.30 15Similarities and contrasts in the structure and function of the calcium transporter ATP2A1 and the copper transporter ATP7B PHYSIOLOGICAL ROLES OF CA2+ AND CU+ ATPASES Ca2+ is a specific activator of muscle fibrils. Acti- vation of contraction depends on Ca2+ delivery and, in turn, and relaxation depends on reduction of Ca2+ con- centration in the cytoplasm of skeletal and cardiac mus- cle cells. At rest, the cytosolic concentration of Ca2+ is much lower than in extracellular fluids and in the intra- cellular vesicles of sarcoplasmic reticulum. Muscle acti- vation occurs when plasma membrane electrical action potentials open passive Ca2+ channels, allowing flux of Ca2+ in the cytosol for activation of myofibrils. Follow- ing the end of action potential, passive channels close, and cytosolic Ca2+ is returned to extracellular fluids and to the sarcoplasmic reticulum interior through active transport by the Ca2+ ATPase. Due to time limits and quantities of Ca2+ available, passive fluxes and active transport across the sarcoplamic reticulum membrane are much prevalent over those across the outer plasma membrane. In the diagram on Fig 13, a cardiac myocyte is shown with the Ca2+ ATPase (ATP) inserted in the plasma membrane (sarcolemma) and the sarcoplasmic reticulum membrane, collecting Ca2+ to induce relaxa- tion, and to be then released upon membrane excitation to induce contraction upon binding to myofibrils.31 The inset shows the time course of an electrical action poten- tial, Ca2+ release, and occurrence of contraction. Chan- nels for passive diffusion of Ca2+, and mitochondria are also shown. Copper is a required metal for homeostasis of plants, bacteria and eukaryotic organisms, determining confor- mation and activity of many metalloproteins and enzyme such as cytochrome oxidase and superoxide dismutase. Furthermore, due to possible reactivity with non-specific proteins and toxic effects, elaborate systems of absorp- tion, concentration buffering, delivery of specific protein sites and elimination, require a complex system including small carriers, chaperones and active transporters. The P-type copper ATPases provide and important system for acquisition, active transport, distribution and elimination of copper. A diagram of copper distribution in eukaryot- Figure 11. Diagrammatic comparison of the calcium and copper ATPases, showing the sequential conformational transitions of the catalytic and transport cycle. The two transported calcium ions exit the ATPase from the E2P state, and the ion exit pathway closes con- comitantly to hydrolytic cleavage of Pi and transition to the E2.Pi state. On the other hand, the copper ions exit the ATPase from the E2P state, but the exit pathway remains open in the E2.Pi state, and closes only in the E2 state after release of Pi.30 Figure 12. Intracellular distribution of ATP7b in COS1 cells expressing WT enzyme (A), subjected to mutation at Asp-1027 (B), Ser-478, Ser-481, Ser-1121 and SER-1453 (C), at the transmem- brane (TMBD) copper site (D), or at the sixth NMBD copper site (E). Note the presence of cytosolic trafficking vesicles with WT enzyme (A), and even and even following Asp-1027 mutation (B), but no trafficking following serine, NMBD or TMBD copper sites (C, D and E).25, 29 16 Giuseppe Inesi ic cells is given in Fig. 14, where it is shown that copper is imported into the cells copper permeases (Ctr1: oval cell membrane bound circles).32 Incoming Cu2+ does not remain free in the cyto- sol, but is rather bound to various chaperones deliver- ing it to specific proteins and secretory pathway. The Cu2+ ATPase (Ccc2 in the figure with the trans-Golgi- Network) binds Cu2+ through the intervention of the Atx1 chaperone, for delivery and transport across the cell membrane, or other destination depending on cell specificity. Cu2+ delivery to the cytochrome c oxidase complex (CcO) involves Cox11, Sco1 and Cox 17 chaper- ones. Nuclear encoded chaperone proteins are imported unfolded across the mitochondrial membrane by a trans- locase, and then acquired in the inner mitochondrial space following introduction of disulphide bonds with the intervention of specific coupled enzyme. In summary, it is evident that Ca2+ and Cu2+ ATPas- es are indispensable components of physiological sys- tems, and the chemistry of their catalytic and transport mechanism is linked to biological function. Transport ATPases are required to regulate the concentrations of Ca2+ and Cu2+ within cells and cellular compartments, utilizing the energy of ATP to sustain appropriate con- centrations across membranes. Appropriate cation con- centrations are required to activate specific enzymes in one direction, and to produce relaxation and avoid toxic consequences in the other direction. REFERENCES 1. R.W. Albers, Ann. Rev. Biochem. 1967, 36, 727-756. 2. R.L. Post, C. Hegyvary, S. Kume, J. Bio. Chem. 1972, 247, 6530. 3. L. de Meis, A. 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