Calorimetric studies of ligand-induced modulation of calcium adenosine 5'-triphosphatase from sarcoplasmic reticulum

Epstein, Michael; Kuriki, Yoshitaka; Biltonen, Rodney L.; Racker, Efraim

doi:10.1021/bi00565a016

Cited by 31 publications

(24 citation statements)

References 26 publications

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“…Thus, binding can be weak when the ligand has in fact a very large effect on the properties of the protein. Very similar numerical data have been reported, for instance, for the binding of Mg 2+ to a Ca 2+ ATPase from sarcoplasmic reticulum (Epstein et al 1980): K298 = 143/M; AH~ = -318 kJ/mol; AS~ = -1025 J/tool 9 K. Such values do not, however, have a general significance. In the reported data for ligand-protein interactions, specific instances can be found for very diverse AH ~ and AS ~ values.…”

Section: Discussionsupporting

confidence: 80%

CaEGTA uncompetitively inhibits calcium activation of whorl morphogenesis inAcetabularia

et al. 1997

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Summary.The spacing between adjacent hairs in vegetative whorls of Acetabularia acetabulum (formerly A. mediterranea) was earlier reported as being quantitatively responsive to calcium ion concentration in the culture medium. We here report a quantitative response to the concentration of the calcium-chelator EGTA, in the opposite sense to the effect of calcium. (Increasing [Ca 2+] diminishes the spacing; increasing [EGTA] increases it.) The earlier work was interpreted in terms of control of the spacing by a putative reaction-diffusion mechanism in the cell membrane, in which a receptor R was activated by calcium-binding to initiate the process. We extend this interpretation by treating CaEGTA as an uncompetitive inhibitor of the effect of calcium on R. This leads to thermodynamic constants for CaEGTA binding to the CaR complex: A/-/~ =-250 _+ 60 kJ/mol; AS~ = -820 -+ 200 J/mol 9 K. Consistency of the concentration and temperature dependences reported here with the postulated dynamic mechanism increases the probability that this mechanism is correct.

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Section: Discussionsupporting

confidence: 80%

CaEGTA uncompetitively inhibits calcium activation of whorl morphogenesis inAcetabularia

et al. 1997

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“…The hydrolysis ofATP proceeds with the formation ofa phosphoenzyme (18), and formation of the phosphoenzyme produces a large conformational change. The hydrolysis of the phosphoenzyme is accelerated by Mg2+, and the binding of Mg21 to the enzyme has been extensively studied (19,20 (26). These kinetic results are consistent either with the presence ofan allosteric ATP site or with a single ATP site having different affinities for two conformations of the enzyme (23).…”

Section: Ca'+mg2+-atpasementioning

confidence: 81%

“…The hydrolysis ofATP proceeds with the formation ofa phosphoenzyme (18), and formation of the phosphoenzyme produces a large conformational change. The hydrolysis of the phosphoenzyme is accelerated by Mg2+, and the binding of Mg21 to the enzyme has been extensively studied (19,20). Steady-state kinetics ofATP hydrolysis show apparent negative cooperativity with Hill coefficients of 0.2-0.8, depending on the pH and concentrations of Na+, K+, and Mg2+ (cf.…”

Section: Ca'+mg2+-atpasementioning

confidence: 99%

Unifying concept for the coupling between ion pumping and ATP hydrolysis or synthesis.

Hammes¹

1982

Proc. Natl. Acad. Sci. U.S.A.

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A mechanism is proposed for the coupling between ion transport and enzyme catalysis. The basic concept is that enzymes associated with transport exist in two possible conformations. Each conformation has the potential of catalyzing the enzymatic reaction, and pumping is associated with the conversion of one conformational form to the other. The conformational transition is triggered by the kinetic blockage of specific mechanistic steps for each conformation. Such blockages can cause a cycling between the two conformations concomitant with catalysis. This mechanistic concept is consistent with a variety of results obtained with the Na+,K+-ATPase, the Ca2+,Mg2+-ATPase, and ATP synthesizing enzymes (coupling factors).The association ofATP hydrolysis and synthesis with ion pumping processes is an accepted tenet of modern biochemistry. However, the detailed enzyme mechanism that couples ATP hydrolysis/synthesis and ion pumping is not well understood.The molecular structure and kinetics of several ion-pumping enzymes have been extensively studied. The purpose of this communication is to show that a single mechanistic concept can be used to understand the coupling between ion transport and catalysis for a diverse group of enzymes. Specifically, this concept is that all of these enzymes exist in two possible conformations and that pumping is associated with the conversion of one conformational form to the other. This conformational transition occurs because specific mechanistic steps are blocked for each conformation. Such a mechanism accounts for a variety of complex kinetic observations. A briefreview ofsome ofthe specific enzymes is first presented. The general mechanism then is proposed and discussed. Na',K+-ATPaseFor a recent review ofthis enzyme see ref.1. The enzyme transports K+ into the cell and Na+ out of the cell, both against concentration gradients. Apparently 3 Na+ and 2 K+ are pumped for each ATP hydrolyzed inside the cell (2). The enzyme contains two types ofpolypeptides in equal amounts with molecular weights ofapproximately 105,000 and 40,000 (3). A phosphoryl enzyme is formed by ATP, and an aspartyl residue on the larger polypeptide is phosphorylated (4). Phosphorylation of the enzyme by MgATP requires Na+, whereas dephosphorylation requires K+. Equal binding stoichiometries have been found for tightly bound ATP (5-7), phosphate (5), ouabain (5, 7), and vanadate (7). Probably one site per large polypeptide is present (8). Both ouabain and vanadate are potent inhibitors ofcatalytic activity. Approximately three Na+ binding sites are present per phosphorylation site (9) and two K+ sites are present per ouabain binding site (10). The Na+ binds tightly to the inside and K+ binds tightly to the outside ofthe cell. Both Na' and K+ may bind at the same location, but the access to the binding sites may be different for different ions and different enzyme conformations. Kinetic studies ofATP hydrolysis indicate apparent negative cooperativity with respect to MgATP (cf. refs. 1 and 11). Both negative c...

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“…Mechanism I fit the data at pH 6 in 15% Me 2 SO (8) best, and Mechanism IV fit the data at pH 7 in aqueous solution (3) better than any of the other mechanisms. Organic solvent must be more important than pH in the change to Mechanism I (8), because the pH varied from 6 to 7 in the studies of calcium pump in water that concluded Mg 2ϩ and P i bind randomly (3)(4)(5)(6)(7). However, the shift in half-maximum P i concentration with [Mg 2ϩ ] o , predicted by assuming Mg⅐P i is the substrate in organic solvent (Table I, Mechanism I, Row 2), was not observed when phosphorylation data for expressed wildtype Ca-ATPase at pH 6 in 30% Me 2 SO was plotted against [P i ] o in a more recent study (9).…”

Section: Figmentioning

confidence: 99%

“…A recent review article (2) states categorically that Mg 2ϩ and P i bind randomly to Ca-ATPase. Most published studies of purified enzyme in aqueous solution support this interpretation (3)(4)(5)(6)(7). However, a study in an organic solventwater mixture concluded that the true substrate is Mg⅐P i (8), and recently the equation for the half-maximum Mg⅐P i concentration was used to interpret an increased half-maximum P i concentration when the upstream threonine in a signature amino acid sequence for P-type pumps (DKTGT) was changed to serine (9).…”

mentioning

confidence: 91%

Evidence Calcium Pump Binds Magnesium before Inorganic Phosphate

Nagy

Kane

Tran

et al. 2005

Journal of Biological Chemistry

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Calcium pump-catalyzed18 O exchange between inorganic phosphate and water was studied to test the hypothesis that all P-type pumps bind Mg 2؉ before P i and validate utilization of the rate equation for ordered binding to interpret differences between site-directed mutants and wild-type enzyme. The results were remarkably similar to those obtained earlier with sodium pump (Kasho, V. N., Stengelin, M., Smirnova, I. N., and Faller, L. D. (1997) Biochemistry 36, 8045-8052). The equation for ordered binding of Mg 2؉ before P i fit the data best with only a slight chance (0.6%) of P i binding to apoenzyme. Therefore, P i is the substrate, and Mg 2؉ is an obligatory cofactor. The intrinsic Mg 2؉ dissociation constant from metalloenzyme (K M ‫؍‬ 3.5 ؎ 0.3 mM) was experimentally indistinguishable from the sodium pump value. However, the half-maximal concentration for P i binding to metalloenzyme (K P ‫؍‬ 6.3 ؎ 0.6 mM) was significantly higher (ϳ6-fold), and the probability of calcium pump forming phosphoenzyme from bound P i (P c ‫؍‬ 0.04 ؎ 0.03) was significantly lower (ϳ6-fold) than for the sodium pump. From estimates of the rate constants for phosphorylation and dephosphorylation, the calcium pump appears to catalyze phosphoryl group transfer less efficiently than the sodium pump. Ordered binding of Mg 2؉ before P i implies that both calcium pump and sodium pump form a ternary enzyme⅐metal⅐phos-phate complex, consistent with molecular structures of other haloacid dehalogenase superfamily members that were crystallized with Mg 2؉ and phosphate, or a phosphate analogue, bound.The calcium and sodium pumps are classified as P-type, 1 because the energy required for generating ion gradients across cell membranes is derived from ATP by catalyzing hydrolysis in two, Mg 2ϩ -dependent steps (1). The ␥-phosphoryl group is initially transferred to an aspartyl side chain of the protein in one conformation (E 1 ) forming a covalent phosphoenzyme intermediate. Hydrolysis is completed after a conformational change by transferring the phosphoryl group to water. The second step can be reversed by reacting the second conformation (E 2 ), with P i and can be followed either by radioactive 32 P incorporation into the enzyme, or by stable oxygen isotope ( 18 O) exchange between P i and H 2 O. Phosphorylation by P i is a partial reaction that can be uncoupled from other reactions in the pump cycle experimentally, so that the mechanism is simple enough for derivation of the rate equation. Therefore, measurements combining either 32 P incorporation or 18 O exchange with site-directed mutagenesis are potentially a powerful way of identifying amino acids essential for catalyzing phosphoryl group transfer and of learning their function from estimates of intrinsic constants for interaction of reactants with the enzyme.The problem considered in this report is the mechanism of Mg 2ϩ -dependent phosphorylation by P i . The solution is important because the interpretation of observed differences between mutants and wild-type enzyme depends upon the...

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Calorimetric studies of ligand-induced modulation of calcium adenosine 5'-triphosphatase from sarcoplasmic reticulum

Cited by 31 publications

References 26 publications

CaEGTA uncompetitively inhibits calcium activation of whorl morphogenesis inAcetabularia

CaEGTA uncompetitively inhibits calcium activation of whorl morphogenesis inAcetabularia

Unifying concept for the coupling between ion pumping and ATP hydrolysis or synthesis.

Evidence Calcium Pump Binds Magnesium before Inorganic Phosphate

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