Modulatory ATP binding affinity in intermediate states of E2P dephosphorylation of sarcoplasmic reticulum Ca2+-ATPase

The mechanism of ATP modulation of E2P dephosphorylation of sarcoplasmic reticulum Ca(2+)-ATPase wild type and mutant forms was examined in nucleotide binding studies of states analogous to the various intermediates of the dephosphorylation reaction, obtained by binding of metal fluorides, vanadate, or thapsigargin. Wild type Ca(2+)-ATPase displays an ATP affinity of 4 μM for the E2P ground state analog, 1 μM for the E2P transition state and product state analogs, and 11 μM for the E2 dephosphoenzyme. Hence, ATP binding stabilizes the transition and product states relative to the ground state, thereby explaining the accelerating effect of ATP on dephosphorylation. Replacement of Phe(487) (N-domain) with serine, Arg(560) (N-domain) with leucine, or Arg(174) (A-domain) with alanine or glutamate reduces ATP affinity in all E2/E2P intermediate states. Alanine substitution of Ile(188) (A-domain) increases the ATP affinity, although ATP acceleration of dephosphorylation is disrupted, thus indicating that the critical role of Ile(188) in ATP modulation is mechanistically based rather than being associated with the binding of nucleotide. Mutants with alanine replacement of Lys(205) (A-domain) or Glu(439) (N-domain) exhibit an anomalous inhibition by ATP of E2P dephosphorylation, due to ATP binding increasing the stability of the E2P ground state relative to the transition state. The ATP affinity of Ca(2)E2P, stabilized by inserting four glycines in the A-M1 linker, is similar to that of the E2P ground state, but the Ca(2+)-free E1 state of this mutant exhibits 3 orders of magnitude reduction of ATP affinity.     

Importance of conserved N-domain residues Thr441, Glu442, Lys515, Arg560, and Leu562 of sarcoplasmic reticulum Ca2+-ATPase for MgATP binding and subsequent catalytic steps. Plasticity of the nucleotide-binding site

Nine single mutations were introduced to amino acid residues Thr441, Glu442, Lys515, Arg560, Cys561, and Leu562 located in the nucleotide-binding domain of sarcoplasmic reticulum Ca2+-ATPase, and the functional consequences were studied in a direct nucleotide binding assay, as well as by steady-state and transient kinetic measurements of the overall and partial reactions of the transport cycle. Some partial reaction steps were also examined in mutants with alterations to Phe487, Arg489, and Lys492. The results implicate all these residues, except Cys561, in high affinity nucleotide binding at the substrate site. Mutations Thr441 --> Ala, Glu442 --> Ala, and Leu562 --> Phe were more detrimental to MgATP binding than to ATP binding, thus pointing to a role for these residues in the binding of Mg2+ or to a difference between the interactions with MgATP and ATP. Subsequent catalytic steps were also selectively affected by the mutations, showing the involvement of the nucleotide-binding domain in these reactions. Mutation of Arg560 inhibited phosphoryl transfer but enhanced the E1PCa2 --> E2P conformational transition, whereas mutations Thr441 --> Ala, Glu442 --> Ala, Lys492 --> Leu, and Lys515 --> Ala inhibited the E1PCa2 --> E2P transition. Hydrolysis of the E2P phosphoenzyme intermediate was enhanced in Glu442 --> Ala, Lys492 --> Leu, Lys515 --> Ala, and Arg560 --> Glu. None of the mutations affected the low affinity activation by nucleotide of the phosphoenzyme-processing steps, indicating that modulatory nucleotide interacts differently from substrate nucleotide. Mutation Glu442 --> Ala greatly enhanced reaction of Lys515 with fluorescein isothiocyanate, indicating that the two residues form a salt link in the native protein.     

ATP-binding modes and functionally important interdomain bonds of sarcoplasmic reticulum Ca2+-ATPase revealed by mutation of glycine 438, glutamate 439, and arginine 678

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 --> E(2)P and E(2)P --> E(2) reactions) and Ca(2+) binding (E(2) --> Ca(2)E(1)). In some of the Ca(2+)-ATPase crystal structures, Arg(678) and Glu(439) seem to be involved in the binding of nucleotide or an associated Mg(2+) ion. We have replaced Arg(678), Glu(439), and Gly(438) with alanine to examine their importance for the enzyme cycle and the modulatory effects of ATP and MgATP. The results point to the key role of Arg(678) in nucleotide binding and to the importance of interdomain bonds Glu(439)-Ser(186) and Arg(678)-Asp(203) in stabilizing the E(2)P and E(2) intermediates, respectively. Mutation of Arg(678) had conspicuous effects on ATP/MgATP binding to the E(1) form and ADP binding to 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 --> E(2)P transition were small, suggesting that the nucleotide that accelerates Ca(2)E(1)P --> E(2)P binds differently from that modulating the E(2)P --> E(2) and E(2) --> 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 --> E(2) and acceleration of the latter reaction, indicating that ATP normally modulates E(2)P --> 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).     

Functional role of "N" (nucleotide) and "P" (phosphorylation) domain interactions in the sarcoplasmic reticulum (SERCA) ATPase

Experimental perturbations of the nucleotide site in the N domain of the SR Ca2+ ATPase were produced by chemical derivatization of Lys492 or/and Lys515, mutation of Arg560 to Ala, or addition of inactive nucleotide analogue (TNP-AMP). Selective labeling of either Lys492 or Lys515 produces strong inhibition of ATPase activity and phosphoenzyme intermediate formation by utilization of ATP, while AcP utilization and reverse ATPase phosphorylation by Pi are much less affected. Cross-linking of the two residues with DIDS, however, drastically inhibits utilization of both ATP and AcP, as well as of formation of phosphoenzyme intermediate by utilization of ATP, or reverse phosphorylation by Pi. Mutation of Arg560 to Ala produces strong inhibition of ATPase activity and enzyme phosphorylation by ATP but has a much lower effect on enzyme phosphorylation by Pi. TNP-AMP increases the ATPase activity at low concentrations (0.1-0.3 microM), but inhibits ATP, AcP, and Pi utilization at higher concentration (1-10 microM). Cross-linking with DIDS and TNP-AMP binding inhibits formation of the transition state analogue with orthovanadate. It is concluded that in addition to the binding pocket delimited by Lys 492 and Lys515, Arg560 sustains an important and direct role in nucleotide substrate stabilization. Furthermore, the effects of DIDS and TNP-AMP suggest that approximation of N (nucleotide) and P (phosphorylation) domains is required not only for delivery of nucleotide substrate, but also to favor enzyme phosphorylation by nucleotide and nonnucleotide substrates, in the presence and in the absence of Ca2+. Domain separation is then enhanced by secondary nucleotide binding to the phosphoenzyme, thereby favoring its hydrolytic cleavage.     

Functional consequences of alterations to amino acids located in the hinge domain of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-specific mutagenesis of the sarcoplasmic reticulum Ca(2+)-ATPase was used to investigate the functional roles of 18 amino acid residues located at or near the "hinge-domain," a highly conserved region of the cation-transporting ATPases. Mutation of Lys684 to arginine, alanine, histidine, and glutamine resulted in complete loss of calcium transport function and ATPase activity. For the Lys684----Ala, histidine, and glutamine mutants, this coincided with a loss of the ability to form a phosphorylated intermediate from ATP or Pi. The Lys684----Arg mutant retained the ability to phorphorylate from ATP with normal apparent affinity, demonstrating the importance of the positive charge. On the other hand, no phosphorylation was observed with Pi as substrate in this mutant. Examination of the partial reactions after phosphorylation from ATP in the Lys684----Arg mutant demonstrated a reduction of the rate of transformation of the ADP-sensitive phosphoenzyme intermediate (E1P) to the ADP-insensitive phosphoenzyme intermediate (E2P), which could account for the loss of transport function. Once accumulated, the E2P intermediate was able to decompose rapidly in the presence of K+ at neutral pH. These results may be interpreted in terms of a preferential destabilization of protein phosphate interactions in the E2P form of this mutant. The Asp703----Ala and Asn-Asp707----Ala-Ala mutants were completely inactive and unable to form phosphoenzyme intermediates from ATP or Pi. In these mutants as well as in the Lys684----Ala mutant, nucleotides were found to protect with normal affinity against intramolecular cross-linking induced with glutaraldehyde, indicating that the nucleotide binding site was intact. Mutation of Glu646, Glu647, Asp659, Asp660, Glu689, Asp695, Glu696, Glu715, and Glu732 to alanine did not affect the maximum rates of calcium transport and ATP hydrolysis or the apparent affinities for calcium and ATP. Mutation of the 2 highly conserved proline residues, Pro681 and Pro709, as well as Lys728, to alanine resulted in partially inhibited Ca(2+)-ATPase enzymes with retention of the ability to form a phosphoenzyme intermediate from ATP or Pi and with normal apparent affinities for ATP and calcium. The proline mutants retained the biphasic ATP concentration dependence of ATPase activity, characteristic of the wild-type, and therefore the partial inhibition of turnover could not be ascribed to a disruption of the low affinity modulatory ATP site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Functional consequences of alterations to Thr247, Pro248, Glu340, Asp813, Arg819, and Arg822 at the interfaces between domain P, M3, and L6-7 of sarcoplasmic reticulum Ca2+-ATPase. Roles in Ca2+ interaction and phosphoenzyme processing

Point mutants with alterations to amino acid residues Thr(247), Pro(248), Glu(340), Asp(813), Arg(819), and Arg(822) of sarcoplasmic reticulum Ca(2+)-ATPase were analyzed by transient kinetic measurements. In the Ca(2+)-ATPase crystal structures, most of these residues participate in a hydrogen-bonding network between the phosphorylation domain (domain P), the third transmembrane helix (M3), and the cytoplasmic loop connecting the sixth and the seventh transmembrane helices (L6-7). In several of the mutants, a pronounced phosphorylation "overshoot" was observed upon reaction of the Ca(2+)-bound enzyme with ATP, because of accumulation of dephosphoenzyme at steady state. Mutations of Glu(340) and its partners, Thr(247) and Arg(822), in the bonding network markedly slowed the Ca(2+) binding transition (E2 --> E1 --> Ca(2)E1) as well as Ca(2+) dissociation from Ca(2+) site II back toward the cytosol but did not affect the apparent affinity for vanadate. These mutations may have caused a slowing, in both directions, of the conformational change associated directly with Ca(2+) interaction at Ca(2+) site II. Because mutation of Asp(813) inhibited the Ca(2+) binding transition, but not Ca(2+) dissociation, and increased the apparent affinity for vanadate, the effect on the Ca(2+) binding transition seems in this case to be exerted by slowing the E2 --> E1 conformational change. Because the rate was not significantly enhanced by a 10-fold increase of the Ca(2+) concentration, the slowing is not the consequence of reduced affinity of any pre-binding site for Ca(2+). Furthermore, the mutations interfered in specific ways with the phosphoenzyme processing steps of the transport cycle; the transition from ADP-sensitive phosphoenzyme to ADP-insensitive phosphoenzyme (Ca(2)E1P --> E2P) was accelerated by mutations perturbing the interactions mediated by Glu(340) and Asp(813) and inhibited by mutation of Pro(248), and mutations of Thr(247) induced charge-specific changes of the rate of dephosphorylation of E2P.     

Kinetic characterization of the normal and detergent-perturbed reaction cycles of the sarcoplasmic reticulum calcium pump. Rate-limiting step(s) under different conditions

We previously characterized the structural features of the interaction of sarcoplasmic reticulum membranes with nonsolubilizing concentrations of C12E8, the non-ionic detergent octaethylene glycol monododecyl ether (Andersen, J.P., le Maire, M., Kragh-Hansen, V., Champeil, P., and M?ller, J. V. (1983) Eur. J. Biochem. 134, 205-214). The present study characterizes especially the functional aspects and implications of the detergent-induced perturbation for an understanding of ATPase function. Perturbing detergent decreased Vmax, but left Ca2+ transport intact. Detergent incorporation affected neither the calcium-dependent phosphorylation from ATP, as judged from multimixer quenching experiments, nor the calcium-releasing transition between the two phosphoenzyme forms (Ca2E1P to E2P), as judged from kinetically resolved dual-wavelength measurements with the calcium-sensitive dye antipyrylazo III. However, the decrease in Vmax was accounted for by a decrease in the rate of enzyme dephosphorylation by a factor of 3-4, whereas the Ca2+-dependent transition between the nonphosphorylated enzyme forms (E2 to Ca2E1) was enhanced almost 10-fold. Evidence of a conformational change of E2 by C12E8 toward that of the E1 state to account for the perturbed reactions was obtained from experiments on vanadate reactivity and tryptic degradation pattern. Both direct and steady-state evidence was obtained for an acceleration by ATP of the Ca2E1P to E2P transition which may account for the low affinity modulatory effect of the nucleotide on enzyme turnover. The kinetic data indicated that reduction of ATP hydrolysis by C12E8 coincided with conditions where E2P dephosphorylation becomes rate-limiting (high ATP concentration, low pH, absence of potassium). Otherwise, the Ca2E1P to E2P transition is deduced to be a rate-limiting step for the ATPase cycle, whereas the potential for rate control of the cycle by modulation of the E2 to Ca2E1 transition is very small. Only in special circumstances (absence of potassium, high temperature, and using ITP as a substrate) did this transition become a rate-limiting step, subject to rate enhancement of the whole cycle by detergent perturbation.     

Functional consequences of alterations to amino acids located in the catalytic center (isoleucine 348 to threonine 357) and nucleotide-binding domain of the Ca2+-ATPase of sarcoplasmic reticulum

The sequence of 10 amino acids (ICSDKTGTLT357) at the site of phosphorylation of the rabbit fast twitch muscle Ca2+-ATPase is highly conserved in the family of cation-transporting ATPases. We changed each of the residues flanking Asp351, Lys352, and Thr353 to an amino acid differing in size or polarity and assayed the mutant for Ca2+ transport activity and autophosphorylation with ATP or P1. We found that conservative changes (Ile----Leu, Thr----Ser, Gly----Ala) or the alteration of Cys349 to alanine did not destroy Ca2+ transport activity or phosphoenzyme formation, whereas nonconservative changes (Ile----Thr, Leu----Ser) did disrupt function. These results indicate that very conservative changes in the amino acids flanking Asp351, Lys352, and Thr353 can be accommodated. A number of mutations were also introduced into amino acids predicted to be involved in nucleotide binding, in particular those in the conserved sequences KGAPE519, RDAGIRVIMITGDNK629, and KK713. Our results indicate that amino acids KGAPE519, Arg615, Gly618, Arg620, and Lys712-Lys713 are not essential for nucleotide binding, although changes to Lys515 diminished Ca2+ transport activity but not phosphoenzyme formation. Changes of Gly626 and Asp627 abolished phosphoenzyme formation with both ATP and Pi, indicating that these residues may contribute to the conformation of the catalytic center.     

Functional consequences of alterations to Ile279, Ile283, Glu284, His285, Phe286, and His288 in the NH2-terminal part of transmembrane helix M3 of the Na+,K(+)-ATPase

Mutations Ile279 --> Ala, Ile283 --> Ala, Glu284 --> Ala, His285 --> Ala, His285 --> Lys, His285 --> Glu, Phe286 --> Ala, and His288 --> Ala in transmembrane helix M3 of the Na+,K(+)-ATPase were studied. Except for His285 --> Ala, these mutations were compatible with cell viability, permitting analysis of their effects on the overall and partial reactions of the Na+,K(+)-transport cycle. In Ile279 --> Ala and Ile283 --> Ala, the E1 form accumulated, whereas in His285 --> Lys and His285 --> Glu, E1P accumulated. Phe286 --> Ala displaced the conformational equilibria of dephosphoenzyme and phosphoenzyme in parallel in favor of E2 and E2P, respectively, and showed a unique enhancement of the E1P --> E2P transition rate. These effects suggest that M3 undergoes significant rearrangements in relation to E1-E2 and E1P-E2P conformational changes. Because the E1-E2 and E1P-E2P conformational equilibria were differentially affected by some of the mutations, the phosphorylated conformations seem to differ significantly from the dephospho forms in the M3 region. Mutation of His285 furthermore increased the Na(+)-activated ATPase activity in the absence of K+ ("Na(+)-ATPase activity"). Ile279 --> Ala, Ile283 --> Ala, and His288 --> Ala showed reduced Na+ affinity of the E1 form. The rate of Na(+)-activated phosphorylation from ATP was reduced in Ile279 --> Ala and Ile283 --> Ala, and these mutants showed evidence similar to Glu329 --> Gln of destabilization of the Na(+)-occluded state.     

Functional consequences of alterations to hydrophobic amino acids located at the M4S4 boundary of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-specific mutagenesis was used to investigate the functional roles of amino acids in the relatively hydrophobic sequence Ile-Thr-Thr-Cys-Leu-Ala-320, located at the M4S4 boundary of the sarcomplasmic reticulum Ca(2+)-ATPase. Each of the residues was replaced with either a less hydrophogic, a polar, or a charged residue. Mutants Ile-315----Arg and Leu-319----Arg were devoid of any Ca2+ transport function or ATPase activity, while the mutant Thr-317----Asp retained about 5 and 7% of the wild-type Ca2+ transport and ATPase activities, respectively. These three mutants were able to form the ADP-sensitive phosphoenzyme intermediate (E1P) by reaction with ATP, but this intermediate decayed very slowly to the ADP-insensitive phosphoenzyme intermediate (E2P). In the mutants Ile-315----Arg and Leu-319----Arg, the level of E2P formed in the backward reaction with inorganic phosphate was extremely low, but hydrolysis of E2P occurred at a normal rate. These mutants, in addition, displayed a higher apparent affinity for Ca2+ than the wild-type enzyme. In the mutants Ile-315----Ser and Ile-315----Asp, the Ca2+ transport and ATPase activities were moderately reduced to 30-40% of the wild-type activities, but normal affinities for Ca2+, Pi, and ATP were retained, as was the low affinity modulatory effect of ATP. Mutation of Thr-316 to Asp, Thr-317 to Ala, Cys-318 to Ala and Ala-320 to Arg had little or no effect on Ca2+ transport or ATPase activities. Introduction of two negative and one positive charge by triple mutation of the Ile-Thr-Thr-317 sequence created a mutant enzyme that, although completely inactive, was inserted into the membrane, consistent with a location of these residues on the cytoplasmic side of the M4S4 interface. Our findings suggest that the amphipathic character of the S4 helix and/or the distribution of charges in S4 is important for the stability of the E2P intermediate.     

Mutations of Arg198 in sarcoplasmic reticulum Ca2+-ATPase cause inhibition of hydrolysis of the phosphoenzyme intermediate formed from inorganic phosphate

Arg198 of sarcoplasmic reticulum Ca2+-ATPase was substituted with lysine, glutamine, glutamic acid, alanine, and isoleucine by site-directed mutagenesis. Kinetic analysis was performed with microsomal membranes isolated from COS-1 cells which were transfected with the mutated cDNAs. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme was determined by first phosphorylating the Ca2+-ATPase with 32Pi and then diluting the sample with non-radioactive Pi. This rate was reduced substantially in the mutant R198Q, more strongly in the mutants R198A and R1981, and most strongly in the mutant R198E, but to a much lesser extent in R198K. The reduction in the rate of dephosphorylation was consistent with the observed decrease in the turnover rate of the Ca2+-ATPase accompanied by the steady-state accumulation of the ADP-insensitive phosphoenzyme formed from ATP. These results indicate that the positive charge and high hydrophilicity of Arg198 are critical for rapid hydrolysis of the ADP-insensitive phosphoenzyme.     

Roles of Leu249, Lys252, and Leu253 in membrane segment M3 of sarcoplasmic reticulum Ca2+-ATPase in control of Ca2+ migration and long-range intramolecular communication

Point mutants with alterations to Leu249, Lys252, Leu253, Asp254, and Glu255 in membrane segment M3, and Pro824, Lys825, and Glu826 in loop L6-7, of the sarcoplasmic reticulum Ca2+-ATPase were analyzed functionally by steady-state and transient kinetic methods. In mutants Leu249Ala, Lys252Glu, and Leu253Ala, the rate of Ca2+ dissociation from the cytoplasmically facing high-affinity Ca2+ sites was increased 4- to 7-fold relative to wild type, and in Leu249Ala and Lys252Glu the rate of Ca2+ binding was increased as well. Substitution of Lys252 with arginine, alanine, glutamine, or methionine affected Ca2+ interaction much less, indicating that the negative charge of the glutamate is particularly disturbing. These findings may be understood on the basis of the hypothesis that a water-accessible channel leading between membrane segments M1 and M3 in the thapsigargin-bound Ca2+-free structure [Toyoshima, C., and Nomura, H. (2002) Nature 418, 605-611] is closely related to the migration pathway for Ca2+. The effects of alanine mutations to Leu249 and Leu253 on Ca2+ dissociation may arise from destabilization of the hydrophobic wall lining the pathway. In mutant Lys252Glu, unfavorable interaction between the glutamate and L6-7 may open the pathway. In addition, Leu253Ala, and to a lesser extent some of the other mutations, reduced the rate of the E1PCa2 to E2P transition of the phosphoenzyme, enhanced the rate of dephosphorylation of E2P, and reduced the apparent affinity for vanadate, suggesting interference with the conformational change of the phosphoenzyme and the function of the catalytic site in E2 and E2P.     

Val200 residue in Lys189-Lys205 outermost loop on the A domain of sarcoplasmic reticulum Ca2+-ATPase is critical for rapid processing of phosphoenzyme intermediate after loss of ADP sensitivity

Possible roles of the Lys(189)-Lys(205) outermost loop on the A domain of sarcoplasmic reticulum Ca(2+)-ATPase were explored by mutagenesis. Both nonconservative and conservative substitutions of Val(200) caused very strong inhibition of Ca(2+)-ATPase activity, whereas substitutions of other residues on this loop reduced activity only moderately. All of the Val(200) mutants formed phosphoenzyme intermediate (EP) from ATP. Isomerization from ADP-sensitive EP (E1P) to ADP-insensitive EP (E2P) was not inhibited in the mutants, and a substantially larger amount of E2P actually accumulated in the mutants than in wild-type sarcoplasmic reticulum Ca(2+)-ATPase at steady state. In contrast, decay of EP formed from ATP in the presence of Ca(2+) was strongly inhibited in the mutants. Hydrolysis of E2P formed from P(i) in the absence of Ca(2+) was also strongly inhibited but was faster than the decay of EP formed from ATP, indicating that the main kinetic limitation of the decay comes after loss of ADP sensitivity but before E2P hydrolysis. On the basis of the well accepted mechanism of the Ca(2+)-ATPase, the limitation is likely associated with the Ca(2+)-releasing step from E2P.Ca(2). On the other hand, the rate of activation of dephosphorylated enzyme on high affinity Ca(2+) binding was not altered by the substitutions. In light of the crystal structures, the present results strongly suggest that Val(200) confers appropriate interactions of the Lys(189)-Lys(205) loop with the P domain in the Ca(2+)-released form of E2P. Results further suggest that these interactions, however, do not contribute much to domain organization in the dephosphorylated enzyme and thus would be mostly lost on E2P hydrolysis.     

Effect of phospholipid, detergent and protein-protein interaction on stability and phosphoenzyme isomerization of soluble sarcoplasmic reticulum Ca-ATPase

The purpose of the present study was to elucidate the separate roles of lipid, detergent and protein-protein interaction for stability and catalytic properties of sarcoplasmic reticulum Ca-ATPase solubilized in the non-ionic detergent octa(ethylene glycol) monododecyl ether (C12E8). The use of large-zone high-performance liquid chromatography permitted us to define the self-association state of Ca-ATPase peptide at various detergent, phospholipid and protein concentrations, and also during enzymatic turnover with ATP. Conditions were established for monomerization of Ca-ATPase in the presence of a high concentration of phospholipid relative to detergent. The lipid-saturated monomeric preparation was relatively resistant to inactivation in the absence of Ca2+, whereas delipidated enzyme in monomeric or in oligomeric form was prone to inactivation. Kinetics of phosphoenzyme turnover were examined in the presence and absence of Mg2+. Dephosphorylation rates were sensitive to Mg2+, irrespective of whether the peptide was present in soluble monomeric form or was membrane-bound. C12E8-solubilized monomer without added phospholipid was, however, characterized by a fast initial phase of dephosphorylation in the absence of Mg2+. This was not observed with monomer saturated with phospholipid or with monomer solubilized in myristoylglycerophosphocholine or deoxycholate. The mechanism underlying this difference was shown to be a C12E8-induced acceleration of conversion of ADP-sensitive phosphoenzyme (E1P) to ADP-insensitive phosphoenzyme (E2P). The phosphoenzyme isomerization rate was also found to be enhanced by low-affinity binding of ATP. This was demonstrated both in membrane-bound and in soluble monomeric Ca-ATPase. Our results indicate that a single peptide chain constitutes the target for modulation of phosphoenzyme turnover by Mg2+ and ATP, and that detergent effects, distinct from those arising from disruption of protein-protein contacts, are the major determinants of kinetic differences between C12E8-solubilized and membrane-bound enzyme preparations.     

Functional consequences of alterations to amino acids located in the nucleotide binding domain of the Ca2(+)-ATPase of sarcoplasmic reticulum

Amino acids in three highly conserved segments of the Ca2(+)-ATPase. Asp-Pro-Pro-Arg604, Thr-Gly-Asp627, Thr-Gly-Asp703 as well as Asp707, have been proposed to participate in formation of the nucleotide binding site. We have tested this hypothesis by investigating the properties of mutants with alterations to amino acids within these segments. Most of the mutants were found to be defective in Ca2+ transport function. The inactive mutants could be separated into two classes on the basis of the kinetics of phosphoenzyme intermediate formation and decomposition. One group, Asp601----Asn, Pro603----Leu, Asp627----Glu, and Asp703----Asn, formed phosphoenzyme intermediates with ATP in the presence of Ca2+ and with inorganic phosphate only in the absence of Ca2+, indicating that both the high affinity Ca2+ binding sites and the nucleotide binding sites were intact. In each of these mutants, however, the ADP-sensitive phosphoenzyme intermediate (E1P) decayed to the ADP-insensitive phosphoenzyme intermediate very slowly, relative to the wild-type enzyme. Thus the inability of these mutants to transport Ca2+ was accounted for by an apparent block of the transport reaction at the E1P to E2P conformational transition. Another group, Asp601----Glu, Pro603----Gly, Asp707----Glu, and Asp707----Asn, did not form detectable phosphoenzyme intermediates from either ATP or Pi. Although we have demonstrated an effect on Ca2+ transport of mutations in each of the highly conserved regions predicted to be involved in ATP binding, we cannot yet define their roles in ATP-dependent Ca2+ transport.     

Mutational analysis of the role of Glu309 in the sarcoplasmic reticulum Ca(2+)-ATPase of frog skeletal muscle.

Site-specific mutagenesis was used to analyse the role of the residue, Glu309, in the function of the Ca(2+)-ATPase of frog skeletal muscle sarcoplasmic reticulum by substitution with Ala or Lys. At pH 6.0, 100 microM Ca2+ was unable to prevent phosphorylation from Pi, consistent with previous observations on the Ca(2+)-ATPase of rabbit fast twitch muscle [Clarke, D.M., Loo, T.W, Inesi, G. and MacLennan, D.H. (1989) Nature 339, 476-478]. At neutral pH, however, micromolar concentrations of Ca2+ were sufficient to inhibit phosphorylation of the Glu309----Lys mutant from inorganic phosphate, suggesting that at least one high-affinity Ca2+ site was relatively intact in this mutant. The Glu309----Lys mutant was unable to form a phosphoenzyme from ATP at all Ca2+ concentrations studied (up to 12.5 mM), whereas phosphorylation of the Glu309----Ala mutant occurred at 12.5 mM Ca2+, but not at Ca2+ concentrations in the submillimolar range. Kinetic studies demonstrated a reduced rate of dephosphorylation of the E2P intermediate in the Glu309----Lys mutant. A less pronounced stabilization of E2P was observed with the Glu309----Ala mutant, suggesting a possible role of the charge at the position of Glu309 in phosphoenzyme hydrolysis.     

Presence of a low-affinity nucleotide binding site on the (K+ + H+)-ATPase phosphoenzyme

The effects of Mg2+ and nucleotides on the dephosphorylation process of the (K+ + H+)-ATPase phosphoenzyme have been studied. Phosphorylation with [gamma-32P]ATP is stopped either by addition of non-radioactive ATP or by complexing of Mg2+ with EDTA. The dephosphorylation process is slow and monoexponential when dephosphorylation is initiated with ATP. When phosphorylation is stopped by complexing of Mg2+ the dephosphorylation process is fast and biexponential. The discrepancy could be explained by a nucleotide mediated inhibition of the dephosphorylation process. The I0.5 for ATP for this inhibition is 0.1 mM and that for ADP is 0.7 mM, suggesting that a low-affinity binding site is involved. When Mg2+ is present in millimolar concentrations in addition to the nucleotides the dephosphorylation process is enhanced. Evidence has been obtained that Mg2+ acts through lowering the affinity for ATP. In contrast to K+, Mg2+ does not stimulate dephosphorylation in the absence of nucleotides. Mg2+ and nucleotides show the same interaction in the dephosphorylation process of a phosphoenzyme generated from inorganic phosphate. These findings suggest the presence of a low-affinity nucleotide binding site on the phosphoenzyme, as is found in the (Na+ + K+)-ATPase phosphoenzyme. This low-affinity binding site may function as a feed-back mechanism in proton transport.     

Transient-state kinetics of the ADP-insensitive phosphoenzyme in sarcoplasmic reticulum: implications for transient-state calcium translocation

The kinetics of formation of the ADP-sensitive (EP) and ADP-insensitive (E*P) phosphoenzyme intermediates of the CaATPase in sarcoplasmic reticulum (SR) were investigated by means of the quenched-flow technique. At 21 degrees C, addition of saturating ADP to SR vesicles phosphorylated for 116 ms with 10 microM ATP gave a triphasic pattern of dephosphorylation in which EP and E*P accounted for 33% and 60% of the total phosphoenzyme, respectively. Inorganic phosphate (Pi) release was less than stoichiometric with respect to E*P decay and was not increased by preincubation with Ca2+ ionophore. The fraction of E*P present after only 6 ms of phosphoenzyme formation was similar to that at 116 ms, indicating that isomerization of EP to E*P occurs very rapidly. Comparison of the time course of E*P formation with intravesicular Ca2+ accumulation measured by quenching with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid + ADP revealed that Ca2+ release on the inside of the vesicle was delayed with respect to E*P formation. Since Ca2+ should dissociate rapidly dissociation from the low-affinity transport sites, these results suggest that Ca2+ remains "occluded" after phosphoenzyme isomerization and that a subsequent slow transition controls the rate of Ca2+ release at the intravesicular membrane surface. Analysis of the forward and reverse rate constants for the EP to E*P transition gave an expected steady-state distribution of phosphoenzymes strongly favoring the ADP-insensitive form. In contrast, the observed ratio of EP to E*P was about 1:2. To account for this discrepancy, a mechanism is proposed in which stabilization of the ADP-sensitive phosphoenzyme is brought about by a conformational interaction between adjacent subunits in a dimer.     

Activation of partial reactions of the Ca2+-ATPase from human red cells by Mg2+ and ATP.

(1) At ATP concentrations up to 30 micrometer addition of 0.5 mM MgCl2 in the reaction mixture increases both the rate of formation and the steady-state level of the phosphoenzyme of the Ca2+-ATPase from human red cell membranes. Under these conditions Mg2+ has no effect on the rate of dephosphorylation, which remains slow. (2) In the presence of Mg2+ the rate of dephosphorylation is increased 5 to 10 times by high (1 mM) concentrations of ATP. (3) Provided Mg2+ has reacted with the phosphoenzyme, acceleration of dephosphorylation by ATP takes place in the absence of Mg2+. This suggests that the role of Mg2+ on dephosphorylation is to convert the phosphoenzyme into a form that, after combination with ATP, reacts rapidly with water. (4) The results are consistent with the idea that combination of ATP at a non-catalytic site is needed for rapid dephosphorylation of the Ca2+-ATPase.     

Calcium binding to the H+,K(+)-ATPase. Evidence for a divalent cation site that is occupied during the catalytic cycle

The binding and conformational properties of the divalent cation site required for H+,K(+)-ATPase catalysis have been explored by using Ca2+ as a substitute for Mg2+. 45Ca2+ binding was measured with either a filtration assay or by passage over Dowex cation exchange columns on ice. In the absence of ATP, Ca2+ was bound in a saturating fashion with a stoichiometry of 0.9 mol of Ca2+ per active site and an apparent Kd for free Ca2+ of 332 +/- 39 microM. At ATP concentrations sufficient for maximal phosphorylation (10 microM), 1.2 mol of Ca2+ was bound per active site with an apparent Kd for free Ca2+ of 110 +/- 22 microM. At ATP concentrations greater than or equal to 100 microM, 2.2 mol of Ca2+ were bound per active site, suggesting that an additional mole of Ca2+ bound in association with low affinity nucleotide binding. At concentrations sufficient for maximal phosphorylation by ATP (less than or equal to 10 microM), APD, ADP + Pi, beta,gamma-methylene-ATP, CTP, and GTP were unable to substitute for ATP. Active site ligands such as acetyl phosphate, phosphate, and p-nitrophenyl phosphate were also ineffective at increasing the Ca2+ affinity. However, vanadate, a transition state analog of the phosphoenzyme, gave a binding capacity of 1.0 mol/active site and the apparent Kd for free Ca2+ was less than or equal to 18 microM. Mg2+ displaced bound Ca2+ in the absence and presence of ATP but Ca2+ was bound about 10-20 times more tightly than Mg2+. The free Mg2+ affinity, like Ca2+, increased in the presence of ATP. Monovalent cations had no effect on Ca2+ binding in the absence of ATP but dit reduce Ca2+ binding in the presence of ATP (K+ = Rb+ = NH4 + greater than Na+ greater than Li+ greater than Cs+ greater than TMA+, where TMA is tetramethylammonium chloride) by reducing phosphorylation. These results indicate that the Ca2+ and Mg2+ bound more tightly to the phosphoenzyme conformation. Eosin fluorescence changes showed that both Ca2+ and Mg2+ stabilized E1 conformations (i.e. cytosolic conformations of the monovalent cation site(s)) (Ca.E1 and Mg.E1). Addition of the substrate acetyl phosphate to either Ca.E1 or Mg.E1 produced identical eosin fluorescence showing that Ca2+ and Mg2+ gave similar E2 (extracytosolic) conformations at the eosin (nucleotide) site. In the presence of acetyl phosphate and K+, the conformations with Ca2+ or Mg2+ were also similar. Comparison of the kinetics of the phosphoenzyme and Ca2+ binding showed that Ca2+ bound prior to phosphorylation and dissociated after dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)