Importance of Thr-353 of the Conserved Phosphorylation Loop of the Sarcoplasmic Reticulum Ca2+-ATPase in MgATP Binding and Catalytic Activity
2001; Elsevier BV; Volume: 276; Issue: 38 Linguagem: Inglês
10.1074/jbc.m105434200
ISSN1083-351X
AutoresJohannes D. Clausen, David B. McIntosh, David G. Woolley, Jens Peter Andersen,
Tópico(s)Pancreatic function and diabetes
ResumoMutants in which Thr-353 of the Ca2+-ATPase of sarcoplasmic reticulum had been replaced with alanine, serine, glutamine, cysteine, valine, aspartate, or tyrosine were analyzed functionally. All the mutations severely affected MgATP binding, whereas ATP binding was close to normal in the alanine, serine, glutamine, and valine mutants. In the serine and valine mutants, the maximum rate of phosphorylation from MgATP was 8- and 600-fold lower, respectively, compared with wild type. Replacement of Mg2+ with Mn2+ led to a 1.5-fold enhancement of the maximum phosphorylation rate in the valine mutant and a 5-fold reduction in the wild type. The turnover of the phosphoenzyme formed from MgATP was slowed 1–2 orders of magnitude relative to wild type in the alanine, serine, and valine mutants, but was close to normal in the aspartate and cysteine mutants. Only the serine mutant formed a phosphoenzyme in the backward reaction with Pi, and the hydrolysis of this intermediate was greatly enhanced. Analysis of the functional changes in the mutants in the light of the recent high resolution structure of the Ca2+-ATPase crystallized without the MgATP substrate suggests that, in the native activated state of the enzyme, the side chain hydroxyl of Thr-353 participates in important interactions with nucleotide and phosphate, possibly in catalysis, whereas the main chain carbonyl of Thr-353, but not the side chain, may coordinate the catalytic Mg2+. Mutants in which Thr-353 of the Ca2+-ATPase of sarcoplasmic reticulum had been replaced with alanine, serine, glutamine, cysteine, valine, aspartate, or tyrosine were analyzed functionally. All the mutations severely affected MgATP binding, whereas ATP binding was close to normal in the alanine, serine, glutamine, and valine mutants. In the serine and valine mutants, the maximum rate of phosphorylation from MgATP was 8- and 600-fold lower, respectively, compared with wild type. Replacement of Mg2+ with Mn2+ led to a 1.5-fold enhancement of the maximum phosphorylation rate in the valine mutant and a 5-fold reduction in the wild type. The turnover of the phosphoenzyme formed from MgATP was slowed 1–2 orders of magnitude relative to wild type in the alanine, serine, and valine mutants, but was close to normal in the aspartate and cysteine mutants. Only the serine mutant formed a phosphoenzyme in the backward reaction with Pi, and the hydrolysis of this intermediate was greatly enhanced. Analysis of the functional changes in the mutants in the light of the recent high resolution structure of the Ca2+-ATPase crystallized without the MgATP substrate suggests that, in the native activated state of the enzyme, the side chain hydroxyl of Thr-353 participates in important interactions with nucleotide and phosphate, possibly in catalysis, whereas the main chain carbonyl of Thr-353, but not the side chain, may coordinate the catalytic Mg2+. the sarco(endo)plasmic reticulum Ca2+-transporting adenosine triphosphatase (EC 3.6.1.38) enzyme form with cytoplasmically facing high affinity Ca2+ binding sites enzyme form with low affinity for Ca2+ phosphoenzyme intermediate containing Ca2+ in the occluded state phosphoenzyme intermediate with luminally facing low affinity Ca2+ binding sites N-2-hydroxyethylpiperazine-N′-3-propanesulfonic acid dissociation constant Michaelis constant ligand concentration giving half maximum effect 2-[N-morpholino]ethanesulfonic acid 3-(N-morpholino)propanesulfonic acid N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid 2′,3′-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate A fundamental property of the P-type ion transporting ATPases is their ability to bind the substrate MgATP with high affinity and catalyze the phosphorylation of a conserved aspartic acid residue in the presence of activating ions (1de Meis L. Vianna A.L. Annu. Rev. Biochem. 1979; 48: 275-292Crossref PubMed Scopus (540) Google Scholar). The formation as well as the further processing of the aspartyl phosphorylated intermediate is associated with protein conformational changes that couple the events in the catalytic site with changes in the ion binding sites leading to ion translocation across the membrane (Fig. 1). The recent 2.6-Å resolution structure of the sarcoplasmic reticulum Ca2+-ATPase1crystallized with bound Ca2+, but without the MgATP substrate (2Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1612) Google Scholar), has revealed that the cytoplasmic portion of the protein is made up from three distinct domains (A, P, and N) that are rather loosely attached to each other and must move considerably to accomplish substrate binding and energy transduction (2Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1612) Google Scholar, 3McIntosh D.B. Nat. Struct. Biol. 2000; 7: 532-535Crossref PubMed Scopus (23) Google Scholar). The molecular nature of the binding sites for ATP and the catalytic magnesium ion, as well as the conformational changes involved in energy transduction, are not well understood, and more information is clearly needed about the functions of the individual amino acid residues in the catalytic site. Thr-353 of the sarcoplasmic reticulum Ca2+-ATPase is located in the catalytic region (domain P (2Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1612) Google Scholar)) close to the phosphorylated residue, Asp-351, and is highly conserved within the family of P-type cation pumps. Maruyama and co-workers (4Maruyama K. Clarke D.M. Fujii J. Inesi G. Loo T.W. MacLennan D.H. J. Biol. Chem. 1989; 264: 13038-13042Abstract Full Text PDF PubMed Google Scholar) reported that mutation of Thr-353 to serine or alanine resulted in pumps with reduced Ca2+ transport activity but with preserved ability to undergo phosphorylation. These observations led to the proposal that the main role of Thr-353 is in the events that take place following phosphorylation, i.e. the conformational changes that are required for release of Ca2+ to the lumen or the dephosphorylation (4Maruyama K. Clarke D.M. Fujii J. Inesi G. Loo T.W. MacLennan D.H. J. Biol. Chem. 1989; 264: 13038-13042Abstract Full Text PDF PubMed Google Scholar). More recently, it was shown that Thr-353 is the residue at the phosphorylation site that becomes oxidized by the phosphate transition-state analogue monovanadate upon UV radiation under conditions favoring accumulation of the Ca2+ boundE 1 form (5Hua S. Inesi G. Toyoshima C. J. Biol. Chem. 2000; 275: 30546-30550Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The catalytic site of the P-type ATPases seems to be homologous to that of haloacid dehalogenase, phosphoserine phosphatase, phosphonatase, phosphomutase, and CheY, the response regulator protein of bacterial chemotaxis (6Aravind L. Galperin M.Y. Koonin E.V. Trends Biochem. Sci. 1998; 23: 127-129Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 7Wang W. Kim R. Jancarik J. Yokota H. Kim S.-H. Structure. 2001; 9: 65-71Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 8Ridder I.S. Dijkstra B.W. Biochem. J. 1999; 339: 223-226Crossref PubMed Scopus (105) Google Scholar, 9Morais M.C. Zhang W. Baker A.S. Zhang G. Dunaway-Mariano D. Allen K.N. Biochemistry. 2000; 2000: 10385-10396Crossref Scopus (125) Google Scholar). Upon mutagenesis of the residue at the position equivalent to Thr-353, these proteins show considerable changes in their activity (10Collet J.F. Stroobant V. Pirard M. Delpierre G. Van Schaftingen E. J. Biol. Chem. 1998; 273: 14107-14112Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 11Collet J.F. Stroobant V. Van Schaftingen E. J. Biol. Chem. 1999; 274: 33985-33990Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 12Silversmith R.E. Smith J.G. Guanga G.P. Les J.T. Bourret R.B. J. Biol. Chem. 2001; 276: 18478-18484Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The atomic structures of phosphoserine phosphatase, phosphonatase, and CheY have demonstrated that the main chain carbonyl at the position corresponding to Thr-353 contributes to coordination of the catalytic Mg2+ (7Wang W. Kim R. Jancarik J. Yokota H. Kim S.-H. Structure. 2001; 9: 65-71Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 9Morais M.C. Zhang W. Baker A.S. Zhang G. Dunaway-Mariano D. Allen K.N. Biochemistry. 2000; 2000: 10385-10396Crossref Scopus (125) Google Scholar, 13Bellsolell L. Prieto J. Serrano L. Coll M. J. Mol. Biol. 1994; 238: 489-495Crossref PubMed Scopus (122) Google Scholar, 14Lee S.-Y. Cho H.S. Pelton J.G. Yan D. Henderson R.K. King D.S. Huang L. Kustu S. Berry E.A. Wemmer D.E. Nat. Struct. Biol. 2001; 8: 52-56Crossref PubMed Scopus (148) Google Scholar), and a similar role in Mg2+ coordination has been suggested for the main chain carbonyl of Thr-353 in the P-type ATPases (8Ridder I.S. Dijkstra B.W. Biochem. J. 1999; 339: 223-226Crossref PubMed Scopus (105) Google Scholar). In the present study, we have subjected Thr-353 of the Ca2+-ATPase to mutational analysis to elucidate its role in the catalytic cycle of the pump. Thr-353 has been replaced by serine, alanine, valine, glutamine, aspartate, cysteine, and tyrosine to study the effects of variation in the size, polarity, and charge of the side chain. Several of the reaction steps shown in Fig. 1 were examined, and the results reveal a multiplicity of important functions of the threonine. Oligonucleotide-directed mutagenesis of cDNA encoding the rabbit fast twitch muscle Ca2+-ATPase (SERCA1a isoform) was carried out as described previously (15Vilsen B. Andersen J.P. Clarke D.M. MacLennan D.H. J. Biol. Chem. 1989; 264: 21024-21030Abstract Full Text PDF PubMed Google Scholar). For expression, the wild type or mutant cDNA, inserted in the pMT2 vector (16Kaufman R.J. Davies M.V. Pathak V.K. Hershey J.W. Mol. Cell. Biol. 1989; 9: 946-958Crossref PubMed Scopus (333) Google Scholar), was transfected into COS-1 cells using the calcium phosphate precipitation method (17Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4821) Google Scholar). The microsomal fraction containing expressed wild type or mutant Ca2+-ATPase was isolated by differential centrifugation (18Maruyama K. MacLennan D.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3314-3318Crossref PubMed Scopus (263) Google Scholar). The concentration of expressed Ca2+-ATPase was quantified by a specific enzyme-linked immunosorbent assay (19Vilsen B. Andersen J.P. MacLennan D.H. J. Biol. Chem. 1991; 266: 16157-16164Abstract Full Text PDF PubMed Google Scholar) and by immunoblotting (15Vilsen B. Andersen J.P. Clarke D.M. MacLennan D.H. J. Biol. Chem. 1989; 264: 21024-21030Abstract Full Text PDF PubMed Google Scholar). Expressed wild type SERCA1a, for which the concentration had been determined by measurement of the maximum capacity for phosphorylation by inorganic phosphate in the presence of 30% (v/v) dimethyl sulfoxide (20Sørensen T. Vilsen B. Andersen J.P. J. Biol. Chem. 1997; 272: 30244-30253Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), was used as standard. For evaluation of the expression level, the concentration of expressed Ca2+-ATPase was related to the total microsomal protein concentration determined by the dye-binding method of Bradford (21Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216357) Google Scholar). The ATP-driven transport of45Ca2+ into the microsomal vesicles was measured by filtration, and the ATPase activity was measured by determining the amount of Pi liberated, as described previously (20Sørensen T. Vilsen B. Andersen J.P. J. Biol. Chem. 1997; 272: 30244-30253Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), under conditions corresponding to maximal activity for the wild type at 37 °C, pH 7.0, and 5 mm MgATP. Following the subtraction of the background activity determined with control microsomes isolated from mock-transfected COS-1 cells, the specific activity was calculated by relating the rate of Ca2+ transport or ATP hydrolysis to the expression level. Manual mixing experiments at various buffer and temperature conditions (detailed in the figure legends) were carried out according to the principles described previously (15Vilsen B. Andersen J.P. Clarke D.M. MacLennan D.H. J. Biol. Chem. 1989; 264: 21024-21030Abstract Full Text PDF PubMed Google Scholar, 19Vilsen B. Andersen J.P. MacLennan D.H. J. Biol. Chem. 1991; 266: 16157-16164Abstract Full Text PDF PubMed Google Scholar,20Sørensen T. Vilsen B. Andersen J.P. J. Biol. Chem. 1997; 272: 30244-30253Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Transient kinetic experiments at 25 °C were performed using a Bio-Logic quench-flow module QFM-5 (Bio-Logic Science Instruments, Claix, France) as described previously (22Sørensen T.L. Dupont Y. Vilsen B. Andersen J.P. J. Biol. Chem. 2000; 275: 5400-5408Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In all phosphorylation experiments, acid quenching was performed with 0.5–2 volumes of 25% (w/v) trichloroacetic acid containing 100 mmH3PO4. The acid-precipitated protein was washed by centrifugation and subjected to SDS-polyacrylamide gel electrophoresis in a 7% polyacrylamide gel at pH 6.0 (23Andersen J.P. Vilsen B. Leberer E. MacLennan D.H. J. Biol. Chem. 1989; 264: 21018-21023Abstract Full Text PDF PubMed Google Scholar), and the radioactivity associated with the separated Ca2+-ATPase band was quantified by imaging using a Packard Cyclone Storage Phosphor System. Background phosphorylation levels were subtracted from all data points. The background was usually determined in parallel experiments with control microsomes isolated from mock-transfected COS-1 cells. In some of the dephosphorylation experiments, the constant phosphorylation level reached after the exponential decay was taken as background (maximally 10% of the initial phosphorylation level). The synthesis of [γ-32P]TNP-8N3-ATP, the photolabeling of COS-1 cell microsomes containing wild type or mutant Ca2+-ATPase, the inhibition by ATP, and the quantification of labeled bands by electronic autoradiography following SDS-polyacrylamide gel electrophoresis were carried out as described previously (24Seebregts C.J. McIntosh D.B. J. Biol. Chem. 1989; 264: 2043-2052Abstract Full Text PDF PubMed Google Scholar, 25McIntosh D.B. Woolley D.G. Vilsen B. Andersen J.P. J. Biol. Chem. 1996; 271: 25778-25789Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Generally, the concentration of [γ-32P]TNP-8N3-ATP was 3·K 0.5 in the inhibition experiments with ATP. Details of the buffer composition are given in the figure legends. The ion concentrations in the reaction buffers were calculated using the program WEBMAXC, available on the World Wide Web, and the stability constants therein (26Bers D.M. Patton C.W. Nuccitelli R. Methods Cell Biol. 1994; 40: 3-29Crossref PubMed Scopus (497) Google Scholar). The phosphorylation data were analyzed by nonlinear regression using the SigmaPlot program (SPSS, Inc.). Monoexponential functions were fitted to the phosphorylation and dephosphorylation time courses. Initial phosphorylation rates per ATPase molecule in units of s−1 were obtained from the slopes of the fitted curves at time zero, following normalization of the phosphorylation levels to the enzyme concentration. The analysis of ligand concentration dependences was based on the Hill equation, [EL]=Etot·[L]n/(K0.5n+[L]n)Equation 1 For analysis of the [γ-32P]TNP-8N3-ATP labeling data, a constant or linear component was added to represent nonspecific labeling as described (24Seebregts C.J. McIntosh D.B. J. Biol. Chem. 1989; 264: 2043-2052Abstract Full Text PDF PubMed Google Scholar, 25McIntosh D.B. Woolley D.G. Vilsen B. Andersen J.P. J. Biol. Chem. 1996; 271: 25778-25789Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), and the Hill coefficient was set to 1. The “true” dissociation constant for ATP and MgATP binding was calculated using the previously validated equation for competitive inhibition of the [γ-32P]TNP-8N3-ATP labeling (25McIntosh D.B. Woolley D.G. Vilsen B. Andersen J.P. J. Biol. Chem. 1996; 271: 25778-25789Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Seven mutants with changes to Thr-353 were examined in the present study, Thr-353 → Ala, Thr-353 → Ser, Thr-353 → Gln, Thr-353 → Cys, Thr-353 → Val, Thr-353 → Asp, and Thr-353 → Tyr, all of which were expressed to levels similar to that of the wild type. Under conditions corresponding to maximal activity for the wild type at 37 °C, pH 7.0, 10–100 μm Ca2+, and 5 mm MgATP, mutant Thr-353 → Ser was able to transport Ca2+ and hydrolyze ATP at a rate of ∼20% of the wild type rate, in good agreement with previously published data (4Maruyama K. Clarke D.M. Fujii J. Inesi G. Loo T.W. MacLennan D.H. J. Biol. Chem. 1989; 264: 13038-13042Abstract Full Text PDF PubMed Google Scholar). For all the other Thr-353 mutants, the Ca2+ transport and ATPase activity determined under these conditions constituted less than 5% of the activity corresponding to wild type (data not shown). In Fig. 2 is shown the results of experiments carried out to obtain an initial overview of the phosphorylation properties of the Thr-353 mutants. The wild type Ca2+-ATPase in the Ca2 E 1 state is able to form a phosphoenzyme intermediate by reaction with MgATP (cf. Fig.1). It can be seen in Fig.2 A that only the wild type and the mutant Thr-353 → Ser formed significant amounts of phosphoenzyme upon incubation with 2 μm [γ-32P]ATP for 15 s at 0 °C and pH 7.0 in the presence of 100 μmCa2+ and 5 mm Mg2+ (saturating concentrations for the wild type). When, however, the incubation was carried out for 5 s at 25 °C in the presence of 5 μm [γ-32P]ATP, under otherwise identical conditions, a markedly increased level of phosphorylation was seen for mutants Thr-353 → Ala, Thr-353 → Cys, Thr-353 → Asp, and, in particular, Thr-353 → Val (Fig. 2 B). It has previously been demonstrated that the catalytic Mg2+can be replaced by various other divalent cations. Among these, Mn2+ seems to be the most efficient co-substrate, both for phosphorylation from ATP (27Yamada S. Ikemoto N. J. Biol. Chem. 1980; 255: 3108-3119Abstract Full Text PDF PubMed Google Scholar) and for the backward phosphorylation from Pi (28Mintz E. Lacapere J.J. Guillain F. J. Biol. Chem. 1990; 265: 18762-18768Abstract Full Text PDF PubMed Google Scholar). Titration of the Mn2+ dependence of the ATPase activity has shown that the concentration of Mn2+ required for maximum activity is lower than that of Mg2+ (29Chiesi M. Inesi G. Arch. Biochem. Biophys. 1981; 208: 586-592Crossref PubMed Scopus (47) Google Scholar). Fig. 2 C shows the results of experiments performed under conditions similar to those in Fig.2 B except for the replacement of the 5 mmMg2+ with 0.5 mm Mn2+. This caused a further 2- to 5-fold increase in the phosphorylation level of mutants Thr-353 → Ala, Thr-353 → Cys, Thr-353 → Val, and Thr-353 → Asp, and even mutant Thr-353 → Gln now showed significant phosphorylation. Although Mn2+ forms a tighter complex with sulfur than Mg2+, allowing the use of “Mn2+ rescue” to locate metal ion binding sites (30Piccirilli J.A. Vyle J.S. Caruthers M.H. Cech T.R. Nature. 1993; 361: 85-88Crossref PubMed Scopus (360) Google Scholar, 31Christian E.L. Yarus M. Biochemistry. 1993; 32: 4475-4480Crossref PubMed Scopus (128) Google Scholar), the replacement of Mg2+ with Mn2+ did not increase the phosphorylation level more in Thr-353 → Cys than in Thr-353 → Ala, Thr-353 → Val, or Thr-353 → Asp, suggesting that the side chain does not interact directly with the metal ion. In the absence of Ca2+, the Ca2+-ATPase in theE 2 form can also be phosphorylated in the backward direction of normal turnover with inorganic phosphate, resulting in the accumulation of E 2P,cf. Fig. 1. As seen in Fig. 2 D, all the Thr-353 mutants displayed a severely reduced ability to undergo phosphorylation from 32Pi. Under conditions optimal for phosphorylation from Pi in the wild type, mutant Thr-353 → Ser was phosphorylated to a level of 24% that of the wild type, whereas the remaining six Thr-353 mutants showed no phosphorylation above the level of control microsomes. Contrary to the situation with phosphorylation from ATP, replacement of Mg2+ with Mn2+ did not increase the Pi phosphorylation levels of the mutants. Even Thr-353 → Ser showed no phosphorylation under these conditions, whereas the wild type phosphorylation level remained practically unaltered (data not shown). To investigate the consequences of mutations to Thr-353 for the binding of ATP and MgATP (the latter often being considered the “true substrate” (32Vianna A.L. Biochim. Biophys. Acta. 1975; 410: 389-406Crossref Scopus (137) Google Scholar)), a specific photolabeling assay was used, in which the affinities can be measured by competitive inhibition of [γ-32P]TNP-8N3-ATP photolabeling (24Seebregts C.J. McIntosh D.B. J. Biol. Chem. 1989; 264: 2043-2052Abstract Full Text PDF PubMed Google Scholar, 25McIntosh D.B. Woolley D.G. Vilsen B. Andersen J.P. J. Biol. Chem. 1996; 271: 25778-25789Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar,33McIntosh D.B. Woolley D.G. MacLennan D.H. Vilsen B. Andersen J.P. J. Biol. Chem. 1999; 274: 25227-25236Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The photolabeling is carried out in the absence of Ca2+ (to prevent phosphoryl transfer to the enzyme) and at pH 8.5 (to reduce nonspecific labeling to minimum and ensure that the predominant enzyme conformation is E 1 (33McIntosh D.B. Woolley D.G. MacLennan D.H. Vilsen B. Andersen J.P. J. Biol. Chem. 1999; 274: 25227-25236Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar)), and ATP is added at various concentrations either in the presence or in the absence of Mg2+. Even though the photolabel may bind at a site that differs slightly from that of ATP or MgATP, there is sufficient overlap between the sites to ensure efficient competition, and we previously demonstrated that the competition assay produces highly accurate values for the ATP and MgATP binding affinities of wild type and mutant Ca2+-ATPase expressed in COS-1 cells (25McIntosh D.B. Woolley D.G. Vilsen B. Andersen J.P. J. Biol. Chem. 1996; 271: 25778-25789Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar,33McIntosh D.B. Woolley D.G. MacLennan D.H. Vilsen B. Andersen J.P. J. Biol. Chem. 1999; 274: 25227-25236Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Results obtained with the Thr-353 mutants are summarized in TableI and exemplified by Thr-353 → Ala and Thr-353 → Ser in Fig. 3. For the wild type Ca2+-ATPase, the K 0.5 for TNP-8N3-MgATP photolabeling and the dissociation constant,K D, for MgATP determined in the presence of Mg2+ have previously been shown to be close to 1.0 and 0.5 μm, respectively (25McIntosh D.B. Woolley D.G. Vilsen B. Andersen J.P. J. Biol. Chem. 1996; 271: 25778-25789Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 33McIntosh D.B. Woolley D.G. MacLennan D.H. Vilsen B. Andersen J.P. J. Biol. Chem. 1999; 274: 25227-25236Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Under the same conditions, all the Thr-353 mutants, apart from Thr-353 → Asp, showed concentration dependences for TNP-8N3-MgATP labeling quite similar to that of the wild type, indicating that Thr-353 does not contribute significantly to the binding of the photolabel (Fig. 3 A and Table I). On the other hand, the affinity for MgATP was, for all Thr-353 mutants, severalfold lower (K D increased) than that of the wild type: 6- to 10-fold for mutants Thr-353 → Ser, Thr-353 → Cys, and Thr-353 → Val, 65- to 187-fold for Thr-353 → Ala, Thr-353 → Gln, and Thr-353 → Tyr, and more than 1000-fold for Thr-353 → Asp (Fig. 3 B and Table I).Table INucleotide binding parameters of wild type and mutant Ca2+-ATPases in the presence and absence of Mg2+Mg2+/EGTA1-aMedium: 25 mmEPPS/tetramethyl ammonium hydroxide (pH 8.5), 20% (w/v) glycerol, 1 mm MgCl2, 0.5 mm EGTA.EDTA1-bMedium: 25 mmEPPS/tetramethyl ammonium hydroxide (pH 8.5), 20% (w/v) glycerol, 2 mm EDTA.K 0.5 (TNP-8N3-MgATP)K D(MgATP)1-cThe “true”K D calculated under the assumption of competitive inhibition as previously described (24, 25). In the inhibition experiments, the concentration of TNP-8N3-ATP was 3 ·K 0.5, except for mutant Thr-353 → Asp in the presence of Mg2+ and EGTA, where it was equal to theK 0.5 value.K 0.5 (TNP-8N3-ATP)K D(ATP)1-cThe “true”K D calculated under the assumption of competitive inhibition as previously described (24, 25). In the inhibition experiments, the concentration of TNP-8N3-ATP was 3 ·K 0.5, except for mutant Thr-353 → Asp in the presence of Mg2+ and EGTA, where it was equal to theK 0.5 value.μmWild type0.981-dData from Refs. 25 and 33 are included for comparison.0.541-dData from Refs. 25 and 33 are included for comparison.0.111-dData from Refs. 25 and 33 are included for comparison.181-dData from Refs. 25 and 33 are included for comparison.Thr-353 → Ala0.64350.06010Thr-353 → Ser1.25.50.1020Thr-353 → Gln1.5400.1328Thr-353 → Cys0.703.40.36118Thr-353 → Val1.24.60.1530Thr-353 → Asp76100.58171Thr-353 → Tyr2.01010.4692Photolabeling and inhibition with ATP/MgATP was carried out as described in Refs. 24Seebregts C.J. McIntosh D.B. J. Biol. Chem. 1989; 264: 2043-2052Abstract Full Text PDF PubMed Google Scholar and 25McIntosh D.B. Woolley D.G. Vilsen B. Andersen J.P. J. Biol. Chem. 1996; 271: 25778-25789Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar.1-a Medium: 25 mmEPPS/tetramethyl ammonium hydroxide (pH 8.5), 20% (w/v) glycerol, 1 mm MgCl2, 0.5 mm EGTA.1-b Medium: 25 mmEPPS/tetramethyl ammonium hydroxide (pH 8.5), 20% (w/v) glycerol, 2 mm EDTA.1-c The “true”K D calculated under the assumption of competitive inhibition as previously described (24Seebregts C.J. McIntosh D.B. J. Biol. Chem. 1989; 264: 2043-2052Abstract Full Text PDF PubMed Google Scholar, 25McIntosh D.B. Woolley D.G. Vilsen B. Andersen J.P. J. Biol. Chem. 1996; 271: 25778-25789Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In the inhibition experiments, the concentration of TNP-8N3-ATP was 3 ·K 0.5, except for mutant Thr-353 → Asp in the presence of Mg2+ and EGTA, where it was equal to theK 0.5 value.1-d Data from Refs. 25McIntosh D.B. Woolley D.G. Vilsen B. Andersen J.P. J. Biol. Chem. 1996; 271: 25778-25789Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar and 33McIntosh D.B. Woolley D.G. MacLennan D.H. Vilsen B. Andersen J.P. J. Biol. Chem. 1999; 274: 25227-25236Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar are included for comparison. Open table in a new tab Photolabeling and inhibition with ATP/MgATP was carried out as described in Refs. 24Seebregts C.J. McIntosh D.B. J. Biol. Chem. 1989; 264: 2043-2052Abstract Full Text PDF PubMed Google Scholar and 25McIntosh D.B. Woolley D.G. Vilsen B. Andersen J.P. J. Biol. Chem. 1996; 271: 25778-25789Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar. In the absence of Mg2+ (i.e. presence of EDTA), the wild type displays a 10-fold higher affinity (decrease inK 0.5) for TNP-8N3-ATP labeling as compared with the affinity in the presence of Mg2+. By contrast, the affinity for ATP is 33-fold lower than the affinity for MgATP (33McIntosh D.B. Woolley D.G. MacLennan D.H. Vilsen B. Andersen J.P. J. Biol. Chem. 1999; 274: 25227-25236Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Like the wild type, all mutants showed higher affinity for the photolabel in the absence of Mg2+ than in its presence (Fig. 3 A and Table I). The affinity for ATP determined by competition with the photolabel was for mutant Thr-353 → Cys 35-fold lower than the affinity for MgATP (Table I), whereas mutants Thr-353 → Ser and Thr-353 → Val showed only a 4- to 7-fold decrease in the affinity upon removal of Mg2+ (compare with 33-fold for the wild type), and mutants Thr-353 → Ala, Thr-353 → Gln, Thr-353 → Asp, and Thr-353 → Tyr displayed an affinity for ATP higher than or similar to the affinity for MgATP (Fig. 3 C and Table I). In fact, the ATP affinities of Thr-353 → Ala, Thr-353 → Ser, Thr-353 → Gln, and Thr-353 → Val were rather similar to that of the wild type despite the severalfold lowering of their MgATP affinities. This shows that Thr-353 is important for the binding of MgATP but less so for the binding of ATP. It is furthermore noteworthy that mutant Thr-353 → Asp exhibited the lowest nucleotide affinity of all the Thr-353 mutants under every condition tested, suggesting electrostatic repulsion of the nucleotide by the negatively charged aspartate side chain. The time course of phosphorylation from [γ-32P]ATP was studied for selected Thr-353 mutants under conditions identical to those corresponding to Fig. 2,B and C. In the presence of Mg2+, the wild type reaches the steady-state level of phosphorylation within a few hundred milliseconds, with a slight initial overshoot (Fig.4 A, cf. also Ref.22Sørensen T.L. Dupont Y. Vilsen B. Andersen J.P. J. Biol. Chem. 2000; 275: 5400-5408Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). For simplicity, a monoexponential function was fitted to the data, giving an apparent rate constant k obs = 63 s−1 for the approach to steady state. Under the same conditions, the k obs was 7.3 s−1for Thr-353 → Ser, 0.9 s−1 for Thr-353 → Cys, and only 0.16 s−1 for Thr-353 → Val (Fig. 4, A andB). A very slow rise in the phosphorylation level was also seen for mutant Thr-353 → Ala (k obs = 0.11 s−1, data not shown). It should be noted that thek ob
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