Phosphorylation of chloride-ATPase reconstituted from Aplysia gut

The present study was primarily done to compare cation-ATPase phosphorylation kinetics with an anion-ATPase's phosphorylation kinetics because of the paucity of information in this area. Utilizing a proteolipsomal preparation containing Cl(-)-ATPase from Aplysia gut, it was demonstrated that phosphorylation of this P-type ATPase was absolutely dependent upon Mg(2+). In organic phosphate concentrations directly (P(i)) enhanced phosphoprotein formation in the presence of increasing concentrations of Mg(2+). It was also shown that the calculated rate constant for E(1)-P formation was 26/sec. This approximated E(1)-P rate constant values for other electrogenic, uniport P-type ATPases, and therefore it was concluded from the results that the anion-ATPase phosphorylation kinetics did not greatly differ from cation-ATPase phosphorylation kinetics.     

The Aplysia californica Cl- pump is a P-type ATPase: evidence through inhibition studies

Utilizing a proteoliposomal preparation containing Cl(-)-ATPase from Aplysia californica foregut, it was shown that orthovanodate inhibited Cl(-)-ATPase activity, ATP-dependent Cl- transport, ATP-dependent membrane potential change and ATP-dependent phosphorylation. N-ethylmalemide and p-chloromercurobenzoate also inhibited the Cl- pump biochemical and physiological transport characteristics. However, bafilomycin, azide, N, N'-dicyclohexylcarboiimide (DCCD), and efrapeptin had no effect on the Cl- pump biochemical or physiological characteristics, suggesting that this Cl- pump was a P-type ATPase. It was concluded that this P-type ATPase Cl- pump is the mechanism that is responsible for the net absorptive flux of Cl- in the A. californica foregut.     

Slippage and uncoupling in P-type cation pumps; implications for energy transduction mechanisms and regulation of metabolism

P-type ATPases couple scalar and vectorial events under optimized states. A number of procedures and conditions lead to uncoupling or slippage. A key branching point in the catalytic cycle is at the cation-bound form of E(1)-P, where isomerization to E(2)-P leads to coupled transport, and hydrolysis leads to uncoupled release of cations to the cis membrane surface. The phenomenon of slippage supports a channel model for active transport. Ability to occlude cations within the channel is essential for coupling. Uncoupling and slippage appear to be inherent properties of P-type cation pumps, and are significant contributors to standard metabolic rate. Heat production is favored in the uncoupled state. A number of disease conditions, include ageing, ischemia and cardiac failure, result in uncoupling of either the Ca(2+)-ATPase or Na(+)/K(+)-ATPase.     

The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. II. Kinetic characterization of phosphointermediates

(1) The kinetics of the phosphorylated enzymic intermediates of (Na+ + K+)-ATPase from ox brain, which are formed by incubation of the enzyme with 25 microM AT32P, 150 mM Na+ and 1 mM Mg2+, have been studied in dephosphorylation experiments at 1 degree C. The dephosphorylation of the 32P-labelled enzyme was initiated by addition of either 1 mM unlabelled ATP, 2.5 mM ADP or 1 mM unlabelled ATP + ADP in concentrations from 25 to 1000 microM. (2) In the absence of ADP the dephosphorylation curve was linear in a semilogarithmic plot almost from t = 0, whereas by addition of ADP a biphasic behaviour was obtained. The slope of the slow phase of dephosphorylation was virtually independent of the ADP concentration. (3) The results were analysed by the mathematical equation corresponding to the simplest possible model for the interconversion and breakdown of the phosphointermediates: (formula: see text) where alpha, beta, H and G are functions of all the rate constants and H and G furthermore are functions of the initial values for [E1P] and [E2P]. (4) The analysis confirmed the model and enabled the determination of all the rate constants. (5) k-1 was found to be equal to k'-1 + k"-1 . [ADP] indicating an ADP-independent 'spontaneous' dephosphorylation of E1P. The rate constant for this process was close to that for dephosphorylation of E2P, i.e., k'-1 congruent to k3. Also the value of k"-1 was determined. (6) k3 was found to be at least 10 . k-2. The implication of this for the role of the E1P to E2P transition in the Na+ + K+)-stimulated ATP hydrolysis will be discussed in detail in the following paper (Plesner, I.W., Plesner, L., N?rby, J.G. and Klodos, I. (1981) Biochim. Biophys. Acta 643, 483--494). (7) A refinement of the model, accounting for the effect of Na+ on the steady-state ratio between [E1P] and [E2P] is proposed: (formula: see text). At [Na+] = 150 mM as used here, E1P(Na) and E'1P are assumed to be in rapid equilibrium. (8) Comparison of our results with those of others underlines the general validity of the conclusions of the present paper.     

Inhibition of phosphoglucomutase by vanadate

Phosphoglucomutase is inhibited by a complex formed from alpha-D-glucose 1-phosphate (Glc-1-P) and inorganic vanadate (Vi). Both the inhibition at steady state and the rate of approach to steady state are dependent on the concentrations of both Glc-1-P and Vi. Inhibition is competitive versus alpha-D-glucose 1,6-bisphosphate (Glc-P2) and is ascribed to binding of the 6-vanadate ester of Glc-1-P (V-6-Glc-1-P) to the dephospho form of phosphoglucomutase (E). The inhibition constant for V-6-Glc-1-P at pH 7.4 was determined from steady-state kinetic measurements to be 2 x 10(-12) M. The first-order rate constant for approach to steady state increases hyperbolically with inhibitor concentration. The results are consistent with rapid equilibrium binding of V-6-Glc-1-P to E, with dissociation constant 1 x 10(-9) M, followed by rate-limiting conversion of the E.V-6-Glc-1-P complex to another species, E*.V-6-Glc-1-P, with first-order rate constant 4 x 10(-2)s-1. The rate constant determined for the reverse reaction, conversion of E*.V-6-Glc-1-P to E.V-6-Glc-1-P, is 2.5 x 10(-4)s-1. Formation of E*.V-6-Glc-1-P can also occur via binding of glucose 6-vanadate to the phospho form of phosphoglucomutase (E-P) followed by phosphoryl transfer and rearrangement of the enzyme-product complex.     

Dissection of the functional differences between human secretory pathway Ca2+/Mn2+-ATPase (SPCA) 1 and 2 isoenzymes by steady-state and transient kinetic analyses

Human secretory pathway Ca2+/Mn2+-ATPase (SPCA) 2 encoded by ATP2C2 is only expressed in a limited number of tissues, unlike the ubiquitously expressed SPCA1 pump (encoded by ATP2C1, the gene defective in Hailey-Hailey disease). It has not been determined whether there are significant functional differences between SPCA1 and SPCA2 pump enzymes. Therefore, steady-state and transient kinetic approaches were used to characterize the overall and partial reactions of the Ca2+ transport cycle mediated by the human SPCA2 enzyme upon heterologous expression in HEK-293 cells. The catalytic turnover rate of SPCA2 was found enhanced relative to SPCA1 pumps. SPCA2 displayed a very high apparent affinity for cytosolic Ca2+ (K0.5 = 0.025 microm) in activation of the phosphorylation activity but still 2.5-fold lower than that of SPCA1d. Our kinetic analysis traced both differences to the increased rate characterizing the E1 approximately PCa to E2-P transition of SPCA2. Moreover, the reduced rate of the E2 to E1 transition seems to contribute in determining the lower apparent Ca2+ affinity and the increased sensitivity to thapsigargin inhibition, relative to SPCA1d. SPCA2 also displayed a reduced apparent affinity for inorganic phosphate, which could be explained by the observed enhanced rate of the E2-P dephosphorylation. The insensitivity to modulation by pH and K+ concentration of the constitutively enhanced E2-P dephosphorylation of SPCA2 is similar to SPCA1d and possibly represents a novel SPCA-specific feature, which is not shared by sarco(endo)plasmic reticulum Ca2+-ATPases.     

(Na+ + K+)-ATPase: confirmation of the three-pool model for the phosphointermediates of Na+-ATPase activity. Estimation of the enzyme-ATP dissociation rate constant

The dephosphorylation kinetics of acid-stable phosphointermediates of (Na+ + K+)-ATPase from ox brain, ox kidney and pig kidney was studied at 0 degree C. Experiments performed on brain enzyme phosphorylated at 0 degree C in the presence of 20-600 mM Na+, 1 mM Mg2+ and 25 microM [gamma-32P]ATP show that irrespectively of the EP-pool composition, which is determined by Na+ concentration, all phosphoenzyme is either ADP- or K+-sensitive. After phosphorylation of kidney enzymes at 0 degree C with 1 mM Mg2+, 25 microM [gamma-32P]ATP and 150-1000 mM Na+ the amounts of ADP- and K+-sensitive phosphoenzymes were determined by addition of 1 mM ATP + 2.5 mM ADP or 1 mM ATP + 20 mM K+. Similarly to the previously reported results on brain enzyme, both types of dephosphorylation curves have a fast and a slow phase, so that also for kidney enzymes a slow decay of a part of the phosphoenzyme, up to 80% at 1000 mM Na+, after addition of 1 mM ATP + 20 mM K+ is observed. The results obtained with the kidney enzymes seem therefore to reinforce previous doubts about the role played by E1 approximately P(Na3) as intermediate of (Na+ + K+)-ATPase activity. Furthermore, for both kidney enzymes the sum of ADP- and K+-sensitive phosphoenzymes is greater than E tot. In experiments on brain enzyme an estimate of dissociation rate constant for the enzyme-ATP complex, k-1, is obtained. k-1 varies between 1 and 4 s-1 and seems to depend on the ligands present during formation of the complex. The highest values are found for enzyme-ATP complex formed in the presence of Na+ or Tris+. The results confirm the validity of the three-pool model in describing dephosphorylation kinetics of phosphointermediates of Na+-ATPase activity.     

Ligand-dependent reactivity of (Na+ + K+)-ATPase with showdomycin

Showdomycin inhibited pig brain (Na+ + K+)-ATPase with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na+, K+, Mg2+ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1) Na+ + Mg2+ + ATP, (2) Mg2+, Mg2+ + K+, K+ and none, (3) Na+ + Mg2+, Na+, K+ + Na+ and Na+ + K+ + Mg2+, (4) Mg2+ + K+ + ATP, K+ + ATP and Mg2+ + ATP, (5) K+ + Na + + ATP, Na+ + ATP, Na+ + K+ + Mg2+ + ATP and ATP. The highest rate was obtained in the presence of Na+, Mg2+ and ATP. The apparent concentrations of Na+, Mg2+ and ATP for half-maximum stimulation of inhibition (KS0.5) were 3 mM, 0.13 mM and 4 MicroM, respectively. The rate was unchanged upon further increase in Na+ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E1, E2, ES, E1-P and E2-P, i. e., E2 has higher reactivity with showdomycin than E1, while E2-P has almost the same reactivity as E1-P. We conclude that the reaction of (Na+ + K+)- ATPase proceeds via at least four kinds of enzyme form (E1, E2, E1 . nucleotide and EP), which all have different conformations.     

On the mechanism of activation of the plasma membrane Ca2+-ATPase by ATP and acidic phospholipids

The activation of purified and phospholipid-depleted plasma membrane Ca2+-ATPase by phospholipids and ATP was studied. Enzyme activity increased with [ATP] along biphasic curves representing the sum of two Michaelis-Menten equations. Acidic phospholipids (phosphatidylinositol (PI) and phosphatidylserine (PS)) increased Vmax without affecting apparent affinities of the ATP sites. In the presence of 20 microm ATP, phosphorylation of the enzyme preincubated with Ca2+ (CaE1) was very fast (kapp congruent with 400 s-1). vo of phosphorylation of CaE1 increased with [ATP] along a Michaelis-Menten curve (Km of 15 microm) and was phospholipid-independent. Without Ca2+ preincubation (E1 + E2), vo of phosphorylation was also phospholipid-independent, but was slower and increased with [ATP] along biphasic curves. The high affinity component reflected rapid phosphorylation of CaE1, the low affinity component the E2 --> E1 shift, which accelerated to a rate higher than that of the ATPase activity when ATP was bound to the regulatory site. Dephosphorylation of EP did not occur without ATP. Dephosphorylation increased along a biphasic curve with increasing [ATP], showing that ATP accelerated dephosphorylation independently of phospholipid. PI, but not phosphatidylethanolamine (PE), accelerated dephosphorylation even in the absence of ATP. kapp for dephosphorylation was 57 s-1 at 0 microM ATP; that rate was further increased by ATP. Steady-state [EP] x kapp for dephosphorylation varied with [ATP], and matched the Ca2+-ATPase activity measured under the same conditions. Apparently, the catalytic cycle is rate-limited by dephosphorylation. Acidic phospholipids stimulate Ca2+-ATPase activity by accelerating dephosphorylation, while ATP accelerates both dephosphorylation and the conformational change from E2 to E1, further stimulating the ATPase activity.     

Identification and function of a cytoplasmic K+ site of the Na+, K+ -ATPase

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)].E(2).MgF(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 P- and A-domains in the E(2) state.     

Interaction of a K(+)-competitive inhibitor, a substituted imidazo[1,2a] pyridine, with the phospho- and dephosphoenzyme forms of H+, K(+)-ATPase

Defining the structural and catalytic properties of the ion transport site(s) of enzyme-phosphorylating ATPases is of key importance in understanding the mechanism of ion transport by these enzymes. In the case of the H+, K(+)-ATPase, SCH 28080 (3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)imidazo[1,2a]-pyridine) has been shown to act as a high affinity, extracytosolic, K(+)-competitive inhibitor of Mg2+, K(+)-ATPase activity (Wallmark, B., Briving, C., Fryklund, J., Munson, K., Jackson, R., Mendlein, J., Rabon, E., and Sachs, G. (1987) J. Biol. Chem. 262, 2077-2084). To define the nature of the SCH 28080-binding site in relation to the catalytic cycle of the enzyme, we have investigated the effects of this potential K+ transport site probe on the steady-state and partial reactions of the H+, K(+)-ATPase. In the absence of K+, SCH 28080 inhibits Mg2(+)-ATPase activity with high affinity (apparent Ki = 30 nM). Inhibition is due to K(+)-like prevention of phosphoenzyme formation. SCH 28080 has no effect on Mg2(+)-catalyzed dephosphorylation. SCH 28080, at concentrations less than 0.5 microM, increases the apparent Km for K+ for Mg2+, K(+)-ATPase activity with little effect on the maximum velocity. At higher concentrations of SCH 28080, reversal of inhibition by higher K+ concentrations is not complete, due to inhibition of ATPase activity by high K+. In contrast, SCH 28080 inhibits K(+)-stimulated dephosphorylation by competitively displacing K+ from phosphoenzyme with an extracytosolic conformation of the monovalent cation site (E2P) at low concentrations of SCH 28080 and K+. At higher concentrations, 10 microM SCH 28080 and 50 mM K+, a slowly dephosphorylating complex with both SCH 28080 and K+ bound to E2P may form which represents a small fraction of the total E2P (15-25%). Preincubation of SCH 28080 with E2P completely blocks K(+)-stimulated dephosphorylation, and K+ is unable to reverse this preincubation effect, indicating that the SCH 28080 dissociation rate is at least as slow as K(+)-independent dephosphorylation of E2P. These findings indicate that SCH 28080 inhibits K(+)-stimulated ATPase activity by competing with K+ for binding to E2P and blocking K(+)-stimulated dephosphorylation. In the absence of K+, SCH 28080 has a higher apparent affinity for E2P, but it permits K(+)-independent dephosphorylation. Since the dissociation rate of SCH 28080 from the enzyme is slow, phosphoenzyme formation is prevented by SCH 28080 remaining bound to the extracytosolic conformation of the monovalent cation site, thereby reducing the steady-state level of phosphoenzyme.     

Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase

P4-ATPases define a eukaryotic subfamily of the P-type ATPases, and are responsible for the transverse flip of specific lipids from the extracellular or luminal leaflet to the cytosolic leaflet of cell membranes. The enzymatic cycle of P-type ATPases is divided into autophosphorylation and dephosphorylation half-reactions. Unlike most other P-type ATPases, P4-ATPases transport their substrate during dephosphorylation only, i.e. the phosphorylation half-reaction is not associated with transport. To study the structural basis of the distinct mechanisms of P4-ATPases, we have determined cryo-EM structures of Drs2p-Cdc50p from Saccharomyces cerevisiae covering multiple intermediates of the cycle. We identify several structural motifs specific to Drs2p and P4-ATPases in general that decrease movements and flexibility of domains as compared to other P-type ATPases such as Na⁺/K⁺-ATPase or Ca²⁺-ATPase. These motifs include the linkers that connect the transmembrane region to the actuator (A) domain, which is responsible for dephosphorylation. Additionally, mutation of Tyr380, which interacts with conserved Asp340 of the distinct DGET dephosphorylation loop of P4-ATPases, highlights a functional role of these P4-ATPase specific motifs in the A-domain. Finally, the transmembrane (TM) domain, responsible for transport, also undergoes less extensive conformational changes, which is ensured both by a longer segment connecting TM helix 4 with the phosphorylation site, and possible stabilization by the auxiliary subunit Cdc50p. Collectively these adaptions in P4-ATPases are responsible for phosphorylation becoming transport-independent.     

Mutational effects on conformational changes of the dephospho- and phospho-forms of the Na+,K+-ATPase

Gly263 of the rat kidney Na(+),K(+)-ATPase is highly conserved within the family of P-type ATPases. Mutants in which Gly263 or the juxtaposed Arg264 had been replaced by alanine were expressed at high levels in COS-1 cells and characterized functionally. Titrations of Na(+),K(+), ATP, and vanadate dependencies of Na(+),K(+)-ATPase activity showed changes in the apparent affinities relative to wild-type compatible with a displacement of the E(1)-E(2) conformational equilibrium in favor of E(1). The level of the K(+)-occluded form was reduced in the Gly263-->Ala and Arg264-->Ala mutants, and the rate constant characterizing deocclusion of K(+) or Rb(+) was increased as much as 20-fold in the Gly263-->Ala mutant. Studies of the sensitivity of the phosphoenzyme to K(+) and ADP showed a displacement of the E(1)P-E(2)P equilibrium of the phosphoenzyme in favor of E(1)P, and dephosphorylation experiments carried out at 25 degrees C on a millisecond time scale using a quenched-flow technique demonstrated a reduction of the E(1)P to E(2)P conversion rate in the mutants. Hence, the mutations displaced the conformational equilibria of dephosphoenzyme and phosphoenzyme in parallel in favor of the E(1) and E(1)P forms. The observed effects were more pronounced in the Gly263-->Ala mutant compared with the Arg264-->Ala mutant. Leu332 mutations that likewise displaced the conformational equilibria in favor of E(1) and E(1)P were also studied. Unlike the Gly263-->Ala mutant the Leu332 mutants displayed a wild-type like rate of K(+) deocclusion. Thus, the effect of the Gly263 mutation on the E(1)-E(2) conformational equilibrium seems to be caused mainly by an acceleration of the K(+)-deoccluding step, whereas in the Leu332 mutants the rate of the reverse reaction seems to be reduced.     

Breakdown of Na+/K+-exchanging ATPase phosphoenzymes formed from ATP and from inorganic phosphate during Na+-ATPase activity.

The reactivity towards Na+ and K+ of Na+/K+-ATPase phosphoenzymes formed from ATP and Pi during Na+-ATPase turnover and that obtained from Pi in the absence of ATP, Na+ and K+ was studied. The phosphoenzyme formed from Pi in the absence of cycling and with no Na+ or K+ in the medium showed a biphasic time-dependent breakdown. The fast component, 96% of the total EP, had a decay rate of about 4 s(-1) in K+-free 130 mm Na+, and was 40% inhibited by 20 mm K+. The slow component, about 0.14 s(-1), was K+ insensitive. Values for the time-dependent breakdown of the phosphoenzymes obtained from ATP and from Pi during Na+-ATPase activity were indistinguishable from each other. In K+-free medium containing 130 mm Na+, the decays followed a single exponential with a rate constant of 0.45 s(-1). The addition of 20 mm K+ markedly increased the decays and made them biphasic. The fast components had a rate of approximately 220 s-1 and accounted for 92-93% of the total phosphoenzyme. The slow components decayed at a rate of about 47-53 s(-1). A second group of experiments examined the reactivity towards Na+ of the E2P forms obtained with ATP and Pi when the enzyme was cycling. In both cases, the rate of dephosphorylation was a biphasic function of [Na+]: inhibition at low [Na+], with a minimum at about 5 mm Na+, followed by recovery at higher [Na+]. Although qualitatively similar, the phosphoenzyme formed from Pi showed slightly less inhibition and more pronounced recovery. These results indicate that forward and backward phosphorylation during Na+-ATPase turnover share the same intermediates.     

P-O bond destabilization accelerates phosphoenzyme hydrolysis of sarcoplasmic reticulum Ca2+ -ATPase

The phosphate group of the ADP-insensitive phosphoenzyme (E2-P) of sarcoplasmic reticulum Ca2+ -ATPase (SERCA1a) was studied with infrared spectroscopy to understand the high hydrolysis rate of E2-P. By monitoring an autocatalyzed isotope exchange reaction, three stretching vibrations of the transiently bound phosphate group were selectively observed against a background of 50,000 protein vibrations. They were found at 1194, 1137, and 1115 cm(-1). This information was evaluated using the bond valence model and empirical correlations. Compared with the model compound acetyl phosphate, structure and charge distribution of the E2-P aspartyl phosphate resemble somewhat the transition state in a dissociative phosphate transfer reaction; the aspartyl phosphate of E2-P has 0.02 A shorter terminal P-O bonds and a 0.09 A longer bridging P-O bond that is approximately 20% weaker, the angle between the terminal P-O bonds is wider, and -0.2 formal charges are shifted from the phosphate group to the aspartyl moiety. The weaker bridging P-O bond of E2-P accounts for a 10(11)-10(15)-fold hydrolysis rate enhancement, implying that P-O bond destabilization facilitates phosphoenzyme hydrolysis. P-O bond destabilization is caused by a shift of noncovalent interactions from the phosphate oxygens to the aspartyl oxygens. We suggest that the relative positioning of Mg2+ and Lys684 between phosphate and aspartyl oxygens controls the hydrolysis rate of the ATPase phosphoenzymes and related phosphoproteins.     

Biphasic kinetics of sarcoplasmic reticulum Ca2+-ATPase and the detergent-solubilized monomer

The mechanism of ATP hydrolysis was studied at 0 degrees C and pH 7.5 using purified leaky vesicles of sarcoplasmic reticulum Ca2+-ATPase and enzyme solubilized in monomeric form with high concentrations of octaethylene glycol monododecyl ether (C12E8). The enzyme reaction of membranous Ca2+-ATPase was characterized by an initial burst in the hydrolysis of ATP and modulated by millimolar concentrations of ATP. For detergent-solubilized Ca2+-ATPase no burst and moderate low affinity modulation was observed, but the reaction was activated both at low (phosphorylating) and intermediate (K0.5 = 0.06 mM) ATP concentrations. A study of the partial reactions indicated that the effects of detergent and ATP were attributable to activation of the E1P----E2P transition which was rate-limiting. E32P dephosphorylation of membranous Ca2+-ATPase and the detergent-solubilized monomer comprised both a slow and a rapid component. The inhibitory effect of high Ca2+ was correlated with the development of a dominant contribution of slow phase dephosphorylation and with ATP-induced extra binding of Ca2+ binding which presumably takes place at the phosphorylation site (ECaP). Ca2+ was bound with lesser affinity to detergent-solubilized Ca2+-ATPase but with qualitatively the same characteristics as to membranous ECaP. Either Ca2+ or Mg2+ was required for dephosphorylation, also after detergent solubilization. It is concluded that ATP hydrolysis occurs by the same steps for membranous and monomeric Ca2+-ATPase and involves formation of either EMgP or ECaP as reaction intermediates, leading to biphasic kinetics, which, therefore, cannot be taken as evidence of an oligomeric function of the enzyme.     

Mimicking of K+ activation by double mutation of glutamate 795 and glutamate 820 of gastric H+,K+-ATPase

Six double mutants of Glu(795) and Glu(820) present in transmembrane domains 5 and 6 of the alpha-subunit of rat gastric H(+),K(+)-ATPase were generated and expressed with the baculovirus expression system. Five of the six mutants exhibited an SCH 28080-sensitive ATPase activity in the absence of K(+). The activity levels decreased in the following order: E795Q/E820A > E795Q/E820Q > E795Q/E820D congruent with E795A/E820A > E795L/E820Q. The E795L/E820D mutant possessed no constitutive activity. The relative low ATPase activity of the E795L/E820Q mutant is due to its low phosphorylation rate so that the dephosphorylation step was no longer rate-limiting. The constitutively active mutants showed a much lower vanadate sensitivity than the wild-type enzyme and K(+)-sensitive mutants, indicating that these mutants have a preference for the E(1) conformation. In contrast to the constitutively active single mutants generated previously, the double mutants exhibited a high spontaneous dephosphorylation rate at 0 degrees C compared to that of the wild-type enzyme. In addition, the H(+),K(+)-ATPase inhibitor SCH 28080 increased the steady-state phosphorylation level of the constitutively active mutants, due to the formation of a stable complex with the E(2)-P form. These studies further substantiate the idea that the empty ion binding pockets of some mutants apparently mimic the K(+)-filled binding pocket of the native enzyme.     

The Crystal Structure of the Ca2+-ATPase 1 from Listeria monocytogenes reveals a Pump Primed for Dephosphorylation

Many bacteria export intracellular calcium using active transporters homologous to the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA). Here we present three crystal structures of Ca²⁺-ATPase 1 from Listeria monocytogenes (LMCA1). Structures with BeF₃^- mimicking a phosphoenzyme state reveal a closed state, which is intermediate between the outward-open E2P and the proton-occluded E2-P* conformations known for SERCA. It suggests that LMCA1 in the E2P state is pre-organized for dephosphorylation upon Ca²⁺ release, consistent with the rapid dephosphorylation observed in single-molecule studies. An arginine side-chain occupies the position equivalent to calcium binding site I in SERCA, leaving a single Ca²⁺ binding site in LMCA1, corresponding to SERCA site II. Observing no putative transport pathways dedicated to protons, we infer a direct proton counter transport through the Ca²⁺ exchange pathways. The LMCA1 structures provide insight into the evolutionary divergence and conserved features of this important class of ion transporters.     

Characterization of the phosphoenzyme that is involved in the Ca2+ -Ca2+ exchange catalyzed by the Ca2+ -ATPase of sarcoplasmic reticulum vesicles

The rate of Ca2+ efflux was determined with 45Ca2+ -loaded sarcoplasmic reticulum vesicles (mainly with the light fraction of vesicles) at pH 6.5 and 0 degrees C. The efflux depended on external Ca2+, Mg2+, ATP and ADP, but it was not activated by AMP. The results indicate that the efflux is derived from Ca2+ -Ca2+ exchange mediated by the phosphoenzyme (EP) of membrane-bound Ca2+ -ATPase. EP was formed with Ca2+ -loaded vesicles (light fraction) under similar conditions without added ADP. The subsequent addition of EGTA and ADP induced triphasic EP dephosphorylation. Three species of EP (EP1, EP2, and EP3) were distinguished on the basis of this dephosphorylation kinetics, EP1, EP2, and EP3, corresponding to the first, second, and third phases of the dephosphorylation. Dephosphorylation of EP1 and EP2 resulted in stoichiometric ATP formation, while dephosphorylation of EP3 led to stoichiometric Pi liberation. The rate of Ca2+ efflux was compatible with that of EP2 dephosphorylation, whereas it was much lower than the rate of EP1 dephosphorylation and much higher than the rate of EP3 dephosphorylation. The intravesicular Ca2+ concentration dependence of the rate of EP2 dephosphorylation agreed with that of the rate of Ca2+ efflux. The results suggest that isomerization between EP1 and EP2 is the rate-limiting process in the Ca2+ -Ca2+ exchange and that EP3 is not involved in this exchange.     

Abolishment of proton pumping and accumulation in the E1P conformational state of a plant plasma membrane H+-ATPase by substitution of a conserved aspartyl residue in transmembrane segment 6

The plasma membrane H(+)-ATPase AHA2 of Arabidopsis thaliana, which belongs to the P-type ATPase superfamily of cation-transporting ATPases, pumps protons out of the cell. To investigate the mechanism of ion transport by P-type ATPases we have mutagenized Asp(684), a residue in transmembrane segment M6 of AHA2 that is conserved in Ca(2+)-, Na(+)/K(+)-, H(+)/K(+)-, and H(+)-ATPases and which coordinates Ca(2+) ions in the SERCA1 Ca(2+)-ATPase. We describe the expression, purification, and biochemical analysis of the Asp(684) --> Asn mutant, and provide evidence that Asp(684) in the plasma membrane H(+)-ATPase is required for any coupling between ATP hydrolysis, enzyme conformational changes, and H(+)-transport. Proton pumping by the reconstituted mutant enzyme was completely abolished, whereas ATP was still hydrolyzed. The mutant was insensitive to the inhibitor vanadate, which preferentially binds to P-type ATPases in the E(2) conformation. During catalysis the Asp(684) --> Asn enzyme accumulated a phosphorylated intermediate whose stability was sensitive to addition of ADP. We conclude that the mutant enzyme is locked in the E(1) conformation and is unable to proceed through the E(1)P-E(2)P transition.