Functional Consequences of Alterations to Amino Acids at the M5S5 Boundary of the Ca -ATPase of Sarcoplasmic Reticulum

The roles of the hydrophobic side chains of residues Phe 750 , Ile 761 , Tyr 763 , Leu 764 , and Ile 765 located at the M5S5 boundary of the Ca 2+ -ATPase of sarcoplasmic reticulum were analyzed by site-directed mutagenesis. Substitution of Tyr 763 with glycine resulted in a new phenotypic variant of the Ca 2+ -ATPase that catalyzed a high rate of Ca 2+ -activated ATP hydrolysis without net accumulation of Ca 2+ in the microsomal vesicles. The ATPase activity of the Tyr 763 → Gly mutant displayed characteristics similar to the ATPase activity of the wild-type enzyme measured in the presence of calcium ionophore, and the mutant was able to form the ADP-insensitive phosphoenzyme intermediate. Mutants Phe 750 → Gly, Ile 761 → Gly, Leu 764 → Gly, and Ile 765 → Gly were able to accumulate Ca 2+ . In mutants Leu 764 → Gly and Ile 765 → Gly, the turnover rate was low due to inhibition of dephosphorylation of the ADP-insensitive phosphoenzyme intermediate. On the other hand, mutant Leu 764 → Lys dephosphorylated rapidly. Mutants Phe 750 → Gly and Leu 764 → Lys displayed apparent Ca 2+ affinities that were reduced two and three orders of magnitude, respectively, relative to that of the wild-type. The roles of the hydrophobic side chains of residues Phe 750 , Ile 761 , Tyr 763 , Leu 764 , and Ile 765 located at the M5S5 boundary of the Ca 2+ -ATPase of sarcoplasmic reticulum were analyzed by site-directed mutagenesis. Substitution of Tyr 763 with glycine resulted in a new phenotypic variant of the Ca 2+ -ATPase that catalyzed a high rate of Ca 2+ -activated ATP hydrolysis without net accumulation of Ca 2+ in the microsomal vesicles. The ATPase activity of the Tyr 763 → Gly mutant displayed characteristics similar to the ATPase activity of the wild-type enzyme measured in the presence of calcium ionophore, and the mutant was able to form the ADP-insensitive phosphoenzyme intermediate. Mutants Phe 750 → Gly, Ile 761 → Gly, Leu 764 → Gly, and Ile 765 → Gly were able to accumulate Ca 2+ . In mutants Leu 764 → Gly and Ile 765 → Gly, the turnover rate was low due to inhibition of dephosphorylation of the ADP-insensitive phosphoenzyme intermediate. On the other hand, mutant Leu 764 → Lys dephosphorylated rapidly. Mutants Phe 750 → Gly and Leu 764 → Lys displayed apparent Ca 2+ affinities that were reduced two and three orders of magnitude, respectively, relative to that of the wild-type. INTRODUCTIONThe Ca 2+ -ATPase of sarcoplasmic reticulum belongs to the ion-translocating ATPases of P-type in which the hydrolysis of ATP is coupled to vectorial transport through formation of an acid-stable phosphoryl aspartyl enzyme intermediate that exists in two major conformational states E1P 1The abbreviations used are: E1PADP-sensitive phosphoenzyme intermediateE1conformation with cytoplasmically facing high affinity Ca 2+ -binding sitesE2conformation with low Ca 2+ affinityE2PADP-insensitive phosphoenzyme intermediateM1-M10putative transmembrane segments numbered from the NH2-terminal end of the peptideS1-S5stalk segments connecting M1-M5 with the cytoplasmic domainsTESN-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid. and E2P (De Meis, 1981; Andersen, 1989). Analysis of the amino acid sequence (MacLennan et al., 1985) along with extensive site-directed mutagenesis studies (MacLennan et al., 1992; Andersen and Vilsen, 1992a) and studies of two-dimensional membrane crystals (Taylor et al., 1986; Toyoshima et al., 1993) have concurred in a structural model for the Ca 2+ -ATPase in which the Ca 2+ binding region is located within a cluster of putative transmembrane α-helices, while the catalytic ATP-binding site is made up from residues in the cytoplasmic domains (Fig. 1). The presence in the Ca 2+ -ATPase of distinct structural entities associated with ATP binding and Ca 2+ binding, respectively, has recently been confirmed by studies of chimeric fusion proteins (Sumbilla et al., 1993; N⊘rregaard et al., 1993). Hence, the energy coupling between scalar and vectorial reactions seems to require long distance communication between the cytoplasmic and transmembrane parts of the pump through conformational changes (Andersen and Vilsen, 1992a). The mutational analysis of the Ca 2+ -ATPase has pinpointed the three transmembrane segments M4, M5, and M6 as central to events associated with Ca 2+ binding and occlusion (Clarke et al., 1989; Andersen and Vilsen, 1992b, 1994; Vilsen and Andersen, 1992d). In addition, this region may be involved in the countertransport of protons (Andersen and Vilsen 1992b, 1994; Vilsen and Andersen, 1992c). An important task is also to locate the amino acid residues responsible for maintenance of the normal physiological coupling of ATP hydrolysis to ion transport, but so far no site-directed mutagenesis data have revealed uncoupled mutants of the Ca 2+ -ATPase.In the present study, the roles of the hydrophobic side chains of Phe 750 , Ile 761 , Tyr 763 , Leu 764 , and Ile 765 located at the border between the fifth transmembrane segment M5 and the putative “stalk” helix S5 have been analyzed by substitution of these residues with glycine. In addition, Leu 764 was replaced by lysine to examine the effect of the positive charge on Ca 2+ binding. These residues are totally conserved among the SERCA-type of Ca 2+ -ATPases. Other cation transporting ATPases of P-type contain motifs that are highly similar although not identical, and the aromatic nature of the residue found at the place of the tyrosine is well conserved (Green, 1989).The results of the functional analysis of the mutants support the notion that these residues are located close to the Ca 2+ binding structure and demonstrate a role of Tyr 763 in ensuring the coupling of ATP hydrolysis to Ca 2+ transport. Hence, the Tyr 763 → Gly mutant catalyzed ATP hydrolysis at a high rate in the absence of any net accumulation of Ca 2+ in the vesicles.EXPERIMENTAL PROCEDURESThe construction and expression of mutant cDNAs as well as most of the methods employed in the analysis of enzyme function have previously been described in detail (Maruyama and MacLennan, 1988; Andersen et al., 1989; Andersen and Vilsen, 1992b; Vilsen et al., 1989, 1991; Vilsen and Andersen, 1992b). In brief, mutations were introduced into the rabbit fast twitch muscle Ca 2+ -ATPase cDNA using the site-specific mutagenesis method of Kunkel(1985). The presence of the correct mutation was confirmed by nucleotide sequencing according to Sanger et al.(1977). The entire Ca 2+ -ATPase cDNA containing the desired mutation was cloned into vector pMT2 (Kaufman et al., 1989) for expression in COS-1 cells (Gluzman, 1981). Microsomes were prepared from transfected cells, and the expression of Ca 2+ -ATPase was by a (Andersen et al., Ca 2+ in the was measured by et al., 1989) of of Ca 2+ -ATPase at for on the transport in containing Ca 2+ and of to the of Ca 2+ at the desired according to using for 2+ and Ca 2+ of and and and Vilsen and Andersen, Ca 2+ was used to Ca 2+ were prepared and at to of calcium of the Ca 2+ measured with from cells transfected with vector without the rate of Ca 2+ was from the of the Ca 2+ as a function of in 2+ of the rate of Ca 2+ in the microsomal vesicles. Ca 2+ was measured as described at in the presence of and the turnover rate was as the between the rate of Ca 2+ of microsomal of Ca 2+ of microsomal and the of microsomal The this as of that measured for the wild-type Ca 2+ -ATPase at Ca 2+ . The of the from which the were at for the wild-type and mutant Phe 750 → Gly and for mutant Tyr 763 → wild-type Ca 2+ , mutant Phe 750 → mutant Ile 761 → mutant Tyr 763 → , mutant Leu 764 → , mutant Ile 765 → rate of ATP hydrolysis was measured at by the described by Vilsen et in the presence of and in the used in most were of the calcium and of to the desired of Ca 2+ . The ATPase activity to the Ca 2+ -ATPase was of the rate of ATP hydrolysis measured in the absence of Ca 2+ of was as in Vilsen et the 2+ at and the Ca 2+ at by of was in the presence of Ca 2+ and as described in Vilsen and Andersen turnover were using the of phosphoenzyme from ATP as a of the of enzyme In the these are as of the turnover rate with the wild-type Ca 2+ phosphoenzyme in the presence of were as a function of Ca 2+ acid at The from was at for in the presence of , 2+ , and to the Ca 2+ the rate of dephosphorylation of the E1P phosphoenzyme intermediate in the absence of Ca 2+ accumulation in the of the calcium was to the was by the of with without by acid at were in a to accumulate the E2P phosphoenzyme intermediate (Andersen et al., 1989; Vilsen et al., 1989). 2+ , Ca 2+ , and from to form E2P in the was for at in the presence of 2+ , and the Ca 2+ with was to the apparent affinity of the Ca 2+ -ATPase for (De et al., so that the was close to in the absence of Ca 2+ . the rate of dephosphorylation of E2P from the was to The was by a of the into containing , 2+ , no by acid at microsomal was and to at by of the and by using an by of The was used to the site for of turnover were at three with The data in and are of is 2+ of ATPase The ATPase activity was measured in the presence of calcium as described and in The turnover rate was as the between the rate of ATP of microsomal and the of microsomal The this as of that measured for the wild-type Ca 2+ -ATPase at Ca 2+ and The of Ca 2+ at a of wild-type Ca 2+ , mutant Phe 750 → mutant Tyr 763 → , mutant Leu 764 → , mutant Ile 765 → 2+ of from of the microsomal from cells transfected with mutant wild-type Ca 2+ -ATPase cDNA was as described The containing of Ca 2+ -ATPase were to at The of the were by from from wild-type Ca 2+ , mutant Phe 750 → mutant Tyr 763 → , mutant Leu 764 → , mutant Ile 765 → mutant Leu 764 → and Ca 2+ of expression of mutants and wild-type Ca 2+ -ATPase were by the of Ca 2+ -ATPase present in the by to the of microsomal Mutants Phe 750 → Gly, Ile 761 → Gly, Leu 764 → Gly, and Ile 765 → Gly were to between and that of the wild-type Ca 2+ -ATPase in The expression of mutants Tyr 763 → Gly and Leu 764 → Lys were between and that of the wild-type in to relative expression that of the wild-type were used for the functional described be able to the rate of Ca 2+ transport into the microsomal containing Ca 2+ in the presence of the of of Ca 2+ into in the is to the The of Ca 2+ at the high Ca 2+ the transport the calcium the for Ca 2+ the results of of the Ca 2+ transport turnover of mutants Phe 750 → Gly, Ile 761 → Gly, Tyr 763 → Gly, Leu 764 → Gly, and Ile 765 → Gly, in the presence of In mutant Ile 761 → Gly, the transport rate measured at Ca 2+ and the apparent Ca 2+ affinity were similar to of the wild-type Ca 2+ On the other hand, the transport of mutants Leu 764 → Gly and Ile 765 → Gly were reduced that of the wild-type an of the Ca 2+ affinities of these Phe 750 → Gly was able to transport Ca 2+ at a but this mutant displayed low apparent Ca 2+ affinity was not to Ca 2+ Hence, at high Ca 2+ Ca 2+ crystals are not the but also to a high this have previously been to the Ca 2+ with at Ca 2+ but in the present was found that the high be up to a Ca 2+ of using the Ca 2+ the Ca 2+ are prepared and the Ca 2+ measured at of at as in these the of Ca 2+ transport in mutant Phe 750 → Gly at Ca 2+ up to Ca 2+ . this Ca 2+ the turnover rate to that of the turnover rate of the also that mutant Tyr 763 → Gly was to transport Ca 2+ at a rate at Ca 2+ up to the of of at Ca 2+ in the presence of was not to any Ca 2+ the to microsomal from cells that been with the vector without In addition, the mutant with Leu 764 → was to transport Ca 2+ at Ca 2+ up to 2+ -activated ATPase Ca 2+ -ATPase activity was measured using the with ATP from et al., in the absence and in the presence of the calcium of at Ca 2+ . The of at hydrolysis of In these was no present to Ca 2+ the and is that in the wild-type enzyme as well as in mutant Phe 750 → Gly the ATPase activity of the calcium is the of the by Ca 2+ present at the (De Meis, 1981). a high Ca 2+ -activated ATPase activity was measured with mutant Tyr 763 → Gly, this mutant was found to accumulate Ca 2+ in the presence of as described with the of Ca 2+ accumulation in mutant Tyr 763 → Gly, was no of ATP hydrolysis in this as by the absence of an effect of the calcium (Fig. Hence, in mutant Tyr 763 → Gly the ATPase activity to be uncoupled from Ca 2+ transport. the results of of ATPase turnover in the presence of the calcium at of Ca 2+ . The ATPase turnover rate of mutant Tyr 763 → Gly was similar to that measured for the wild-type Ca 2+ -ATPase in the presence of The apparent Ca 2+ affinity of mutant Tyr 763 → Gly was reduced relative to that of the at Ca 2+ . In mutant Phe 750 → Gly the apparent Ca 2+ affinity from Ca 2+ of ATPase activity was with the data for Ca 2+ of Ca 2+ transport in Mutants Leu 764 → Gly and Ile 765 → Gly displayed low ATPase is that the ATPase activity of mutant Leu 764 → Gly was that of mutant Ile 765 → Gly, while the Ca 2+ activity of mutant Leu 764 → Gly was that of mutant Ile 765 → Gly also as mutant Leu 764 → Gly may have been uncoupled to Leu 764 → Lys was to at a Ca 2+ of of Ca 2+ -activated ATPase ATP hydrolysis was by the described in the presence of and Ca 2+ . The at The in the the were of the calcium was to by Ca 2+ . was to the ATPase activity not to Ca 2+ wild-type Ca 2+ mutant Tyr 763 → mutant Phe 750 → from cells transfected with vector without that microsomal was in this ATPase activity was by of the on at Ca 2+ The for the Tyr 763 → Gly mutant was on that of the and the wild-type Ca 2+ the mutants displayed a with a in the of a role for ATP as an in to role as 1989). In addition, was found that Tyr 763 → Gly, were to inhibition by from ATP and the wild-type Ca 2+ the mutants were able to from ATP the of Ca 2+ was high and from in the absence of Ca 2+ . The ATP of from ATP of the mutants was from that of the with for ATP The Ca 2+ of from ATP is in The uncoupled mutant Tyr 763 → Gly displayed an apparent Ca 2+ affinity that of the wild-type. The apparent Ca 2+ affinities of mutants Leu 764 → Gly and Ile 765 → Gly were also similar to that of the wild-type. In with the results of Ca 2+ of Ca 2+ transport and Ca 2+ -ATPase the apparent Ca 2+ affinity of mutant Phe 750 → Gly in the with ATP was at that of the wild-type. Leu 764 → Lys displayed Ca 2+ affinity in the with a the described of this mutant to transport Ca 2+ and ATP at Ca 2+ of the inhibition by Ca 2+ of the from may on the function of the Ca 2+ (Andersen and Vilsen, 1992b, The results of are in with that the apparent Ca 2+ affinities in the two of were in the of mutant Phe 750 → The apparent Ca 2+ affinity by of Ca 2+ inhibition of in this mutant was to that of the in to the low Ca 2+ affinity in the of Ca 2+ of from the Ca 2+ -ATPase is to two calcium in a and the binding site has to be to from a of the between the apparent Ca 2+ affinities in the two of with mutant Phe 750 → Gly is that Phe 750 is involved in the binding of the calcium but not in the binding of the calcium ion in the sequence (Andersen and Vilsen, 1992b, of the the turnover of mutants Leu 764 → Gly and Ile 765 → Gly were found to be reduced at of Ca 2+ and ATP that were to the that a the have been in these In the normal Ca 2+ -ATPase the phosphoenzyme intermediate is which is able to the phosphoryl to ATP (De Meis, 1981). intermediate the calcium in an with no to the on side of the membrane and et al., 1991; Vilsen and Andersen, 1992a). The of Ca 2+ at the side of the membrane is to be coupled with the to a phosphoenzyme which is The intermediate to be to the phosphoenzyme intermediate in the with (De Meis, 1981). The is by the hydrolysis of the in E2P and the of and the results of in which the of the E1P and E2P phosphoenzyme were was with ATP (Fig. the E1P intermediate in the wild-type (Andersen et al., 1989; Vilsen et al., with to E2P (Fig. the been with the of the phosphoenzyme was in the dephosphorylation in the was of to the by of Ca 2+ and in In the wild-type Ca 2+ the phosphoenzyme of as has previously been (Andersen et al., 1989; Vilsen et al., 1989). in a dephosphorylation from E1P similar to that of the wild-type was with mutants Phe 750 → Gly, Ile 761 → Gly, and Tyr 763 → Gly, with to ATP at a high In mutant Leu 764 → Gly, the rate of of the phosphoenzyme in the presence of without was reduced relative to that of the wild-type. The of dephosphorylation was in mutant Ile 765 → The mutant was also to dephosphorylation in the of the is as with mutant Ile 765 → Gly, the of phosphoenzyme the with the of E2P in The accumulation of a of E2P in mutant Ile 765 → Gly the E1P intermediate in the wild-type enzyme that the high of the phosphoenzyme of mutant Ile 765 → Gly in the was due to a of the dephosphorylation of was confirmed in the in in which the dephosphorylation of E2P was of the with by of the in the mutants Phe 750 → Gly and Ile 761 → Gly dephosphorylated while the dephosphorylation was in mutant Ile 765 → In addition, the dephosphorylation of E2P in mutant Leu 764 → Gly was The to of the glycine at 764 with is also that the rate of dephosphorylation of E2P was reduced in the uncoupled mutant Tyr 763 → of the phosphoenzyme from The were to at and the of the are with was at in the presence of , 2+ , Ca 2+ , of the Ca 2+ and with the was with and for with without for to with was at for in the presence of 2+ , and to the was dephosphorylation was by of an into containing , 2+ , and and acid was the was at for in the presence of 2+ , Ca 2+ , and The was with and for to In the was with without for to In the was with without for by for to the E2P phosphoenzyme intermediate is to the enzyme form that Ca 2+ at the a for the of ATP hydrolysis from Ca 2+ transport in mutant Tyr 763 → Gly be that E1P is to the of mutant Tyr 763 → Gly to form E2P in the from from ATP was in at and in the absence of these E2P as the major intermediate in the wild-type Ca 2+ due to a reduced rate of dephosphorylation of E2P (Andersen et al., 1989; Vilsen et al., 1989). is in and that the E2P phosphoenzyme intermediate was in the Tyr 763 → Gly and Leu 764 → Gly mutants as as by the of phosphoenzyme to and demonstrate that the ADP-insensitive of the phosphoenzyme in the Tyr 763 → Gly mutant a with present mutant Tyr 763 → Gly as a new phenotypic variant of the Ca 2+ -ATPase which is uncoupled in the that ATP at a high rate in a Ca 2+ -activated without any net accumulation of Ca 2+ in the microsomal vesicles. In this from ATP and the rate of hydrolysis of ATP were by Ca 2+ at a Ca 2+ as low as no Ca 2+ accumulation was at Ca 2+ up to the of The of ATPase activity from Ca 2+ accumulation in the Tyr 763 → Gly mutant was by the of an effect of on the ATP uncoupled ATP hydrolysis catalyzed by the Tyr 763 → Gly mutant through formation of an acid-stable phosphoenzyme intermediate and displayed characteristics similar to the ATPase activity of the wild-type Ca 2+ -ATPase with to and In the wild-type dephosphorylation place through the sequence E1P → E2P → was that the Tyr 763 → Gly mutant is able to form the E2P intermediate from E1P (Fig. E2P is the intermediate to Ca 2+ at the (De Meis, 1981; Andersen, 1989; et al., is that the responsible for the of Ca 2+ is in the Tyr 763 → Gly and that the of net accumulation of Ca 2+ by the mutant is due to an rate of of Ca 2+ from the vesicles. an of Ca 2+ be of the of Ca 2+ through the Ca 2+ -ATPase binding of and to the pump (De Meis, 1991; et al., 1992; and was that this be through of in the of the normal (De Meis, the calcium are not by the Tyr 763 → Gly mutant due to a in the of the is that the the calcium from to the cytoplasmic side is in the mutant so that an E1P intermediate with Ca 2+ is The side of Tyr 763 a role in the not the roles of the aromatic side chains in of ion and 1992; 1993). is also that the of the side of the tyrosine to a and of the transmembrane for as a of changes in helix of apparent Ca 2+ affinity of the uncoupled Tyr 763 → Gly mutant as well as the changes in Ca 2+ affinity with mutants Phe 750 → Gly and Leu 764 → Lys are in with the (Fig. that the M5S5 boundary is located in the of the to the Ca 2+ -binding The effect of the lysine side on Ca 2+ binding be by of the calcium mutant Leu 764 → Lys was able to at a normal while the dephosphorylation of the E2P intermediate was in mutants Leu 764 → Gly and Ile 765 → Gly, and so in the The of E2P dephosphorylation the low ATPase activity with mutants Leu 764 → Gly and Ile 765 → similar of dephosphorylation of E2P has been for other mutants with to residues in transmembrane segments M4, M5, and the putative Ca 2+ residues , , and (Andersen and Vilsen, 1992b, 1994; Vilsen and Andersen, et al., 1993). that protons are by the Ca 2+ -ATPase et al., et al., in a similar to the transport by that the role for these residues in the dephosphorylation of E2P be associated with the of the side to form for the to be (Andersen and Vilsen, 1992b). is that the side chains of Leu 764 and Ile 765 are to the dephosphorylation of E2P the binding structure in the conformational long communication with the The of the to of the glycine with lysine be to the of the side of the also that the presence of a side the site a the ATPase activity of mutant Leu 764 → Gly was that of mutant Ile 765 → Gly, while the Ca 2+ was that also mutant Leu 764 → Gly may have been uncoupled to the present has to the hydrophobic residues at the M5S5 boundary as important for the coupling between ATP hydrolysis and Ca 2+ transport, and for the reactions involved in Ca 2+ binding and E2P be important to in the to the for the INTRODUCTIONThe Ca 2+ -ATPase of sarcoplasmic reticulum belongs to the ion-translocating ATPases of P-type in which the hydrolysis of ATP is coupled to vectorial transport through formation of an acid-stable phosphoryl aspartyl enzyme intermediate that exists in two major conformational states E1P 1The abbreviations used are: E1PADP-sensitive phosphoenzyme intermediateE1conformation with cytoplasmically facing high affinity Ca 2+ -binding sitesE2conformation with low Ca 2+ affinityE2PADP-insensitive phosphoenzyme intermediateM1-M10putative transmembrane segments numbered from the NH2-terminal end of the peptideS1-S5stalk segments connecting M1-M5 with the cytoplasmic domainsTESN-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid. and E2P (De Meis, 1981; Andersen, 1989). Analysis of the amino acid sequence (MacLennan et al., 1985) along with extensive site-directed mutagenesis studies (MacLennan et al., 1992; Andersen and Vilsen, 1992a) and studies of two-dimensional membrane crystals (Taylor et al., 1986; Toyoshima et al., 1993) have concurred in a structural model for the Ca 2+ -ATPase in which the Ca 2+ binding region is located within a cluster of putative transmembrane α-helices, while the catalytic ATP-binding site is made up from residues in the cytoplasmic domains (Fig. 1). The presence in the Ca 2+ -ATPase of distinct structural entities associated with ATP binding and Ca 2+ binding, respectively, has recently been confirmed by studies of chimeric fusion proteins (Sumbilla et al., 1993; N⊘rregaard et al., 1993). Hence, the energy coupling between scalar and vectorial reactions seems to require long distance communication between the cytoplasmic and transmembrane parts of the pump through conformational changes (Andersen and Vilsen, 1992a). The mutational analysis of the Ca 2+ -ATPase has pinpointed the three transmembrane segments M4, M5, and M6 as central to events associated with Ca 2+ binding and occlusion (Clarke et al., 1989; Andersen and Vilsen, 1992b, 1994; Vilsen and Andersen, 1992d). In addition, this region may be involved in the countertransport of protons (Andersen and Vilsen 1992b, 1994; Vilsen and Andersen, 1992c). An important task is also to locate the amino acid residues responsible for maintenance of the normal physiological coupling of ATP hydrolysis to ion transport, but so far no site-directed mutagenesis data have revealed uncoupled mutants of the Ca 2+ -ATPase.In the present study, the roles of the hydrophobic side chains of Phe 750 , Ile 761 , Tyr 763 , Leu 764 , and Ile 765 located at the border between the fifth transmembrane segment M5 and the putative “stalk” helix S5 have been analyzed by substitution of these residues with glycine. In addition, Leu 764 was replaced by lysine to examine the effect of the positive charge on Ca 2+ binding. These residues are totally conserved among the SERCA-type of Ca 2+ -ATPases. Other cation transporting ATPases of P-type contain motifs that are highly similar although not identical, and the aromatic nature of the residue found at the place of the tyrosine is well conserved (Green, 1989).The results of the functional analysis of the mutants support the notion that these residues are located close to the Ca 2+ binding structure and demonstrate a role of Tyr 763 in ensuring the coupling of ATP hydrolysis to Ca 2+ transport. Hence, the Tyr 763 → Gly mutant catalyzed ATP hydrolysis at a high rate in the absence of any net accumulation of Ca 2+ in the vesicles.