Ouabain-binding and phosphorylation of (Na+ + K+) ATPase treated with N-ethylmaleimide or oligomycin.

Ouabain-binding and phosphorylation of (Na+ mk+)-ATPase (EC 3.6.1.3) of the plasma membranes from kidney were investigated after treatment with N-ethylmaleimide or oligomycin. Either of these inhibitors brought about the following changes: the phosphoenzyme, formed in the presence of Na+, Mg2+ and ATP became essentially insensitive to splitting by K+ but was split by ADP. One mole of this ADP-sensitive phosphoenzyme bound one mole of ouabain but the enzyme-ouabain complex was less stable than in the native enzyme primarily because the rate of its dissociation increased. Ouabain was bound to the ADP-sensitive phosphoenzyme in the presence of Mg2+ alone and addition of inorganic phosphate enhanced both the rate of formation and the steady-state level of the enzyme-ouabain complex. The inhibitors did not affect the properties of this second type of complex. Both in the native enzyme and in the enzyme treated with the two inhibitors inorganic phosphate enhanced ouabain binding by phosphorylating the active center of the enzyme as shown (a) by mapping the labeled peptides from the enzyme after peptic digestion, (b) by inhibition of this phosphorylation with Na+ and (c) by the 1:1 stoichiometric relation between this phosphorylation and the amount of bound ouabain. Unlike the phosphoenzyme, the binding of ouabain remained sensitive to K+ in the enzyme treated with the inhibitors. K+ slowed ouabain-binding either in the presence of Na+, Mg2+ and ATP or of Mg2+ and inorganic phosphate. A higher concentration of K+ was needed to slow ouabain-binding either in the presence of Na+, Mg2+ and ATP or of Mg2+ and inorganic phosphate. A higher concentration of K+ was needed to slow ouabain-binding than to stimulate dephosphorylation. This finding is interpreted as being an indication of separate sites for K+ on the enzyme: a site(s) with high K+-affinity which stimulates dephosphorylation, another site(s) with moderate K+-affinity which inhibits ouabain-binding. Inhibitors may enhance formation of the ADP-sensitive phosphoenzyme by blocking interaction between K+ and the site(s) with high affinity.     

Heat capacity and entropy changes of the two major isotypes of bullfrog (Rana catesbeiana) parvalbumins induced by calcium binding

The possible structural changes of the two major isotypes (PA1 and PA2) of parvalbumins from bullfrog (Rana catesbeiana) skeletal muscle caused by Ca2+ binding have been analyzed by microcalorimetric titrations. Titrations of the parvalbumins with Ca2+ have been made in both the absence and presence of Mg2+ at pH 7.0 and at 5, 15, and 25 degrees C. The reactions of the parvalbumins with Ca2+ are exothermic in both the presence and absence of Mg2+ and at every temperature. But the contributions of enthalpy and entropy changes are variable; Mg2+-Ca2+ exchange on PA1 at 25 degrees C is driven almost entirely by a favorable enthalpy change, whereas Ca2+ binding to PA2 at 5 degrees C is driven for the most part by a favorable entropy change. The magnitudes of the hydrophobic and internal vibrational contributions to the heat capacity and entropy changes of the parvalbumins on Ca2+ binding and Mg2+-Ca2+ exchange have been estimated by the empirical method of Sturtevant [Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2236-2240]. Although PA1 (beta) and PA2 (alpha) belong to genetically different lineages, the parvalbumins indicate very similar conformational changes to each other on both Ca2+ binding and Mg2+-Ca2+ exchange. On Mg2+-Ca2+ exchange, the vibrational as well as hydrophobic entropy is slightly increased in a parallel manner. In contrast, on Ca2+ binding, the hydrophobic entropy increases but the vibrational entropy decreases. The increase in the hydrophobic entropy indicates the sequestering of nonpolar groups from the surface to the interior of molecules, while the changes in the vibrational entropy suggest that the overall structures are tightened on Ca2+ binding but loosened on Mg2+-Ca2+ exchange.     

Spectral and kinetic studies of phosphate and magnesium ion binding to yeast inorganic pyrophosphatase

Inorganic pyrophosphatase must bind two phosphate molecules in order to catalyze pyrophosphate synthesis. In this report it is shown that Pi causes marked effect on the absorption spectrum of baker's yeast inorganic pyrophosphatase and this effect can be used to analyze Pi binding to this enzyme. A series of absorbance versus Pi concentration curves in the presence of 0.5-20 mM free Mg2+ were obtained at pH 7.2 and computer-fitted to 19 models. The dissociation constant of magnesium phosphate (8.5 +/- 0.4 mM) used in this analysis was measured with a Mg2+-sensitive electrode. The best model implies successive binding of two magnesium phosphate molecules or random-order binding of magnesium phosphate and free phosphate molecules. The first route predominates at physiological concentrations of Mg2+. The Pi-inhibition pattern of pyrophosphate hydrolysis confirmed that Pi adds to the active site and provided further evidence for the existence of an activating Pi-binding site. The possibility is raised that the pathways of pyrophosphate synthesis and hydrolysis by inorganic pyrophosphatase may differ in the sense that the binding of the fourth metal ion/subunit may facilitate the synthesis and inhibit the hydrolysis.     

Studies on (Na+ + K+)-activated ATPase. XXXVIII. A 100 000 molecular weight protein as the low-energy phosphorylated intermediate of the enzyme.

Phosphorylation of NaI-treated bovine brain cortex microsomes by inorganic phosphate in the presence of Mg2+ and ouabain has been studied at 0 degrees C (pH 7.4) and 20 degrees C (pH 7.0). Nearly maximal (90%) and half-maximal phosphorylation are achieved at 20 degrees C within 2 min with 50--155 and 5.6--17 muM 32Pi, respectively, and at 0 degrees C within 75 s with 300--600 and 33--66 muM 32Pi, respectively. Maximal phosphorylation yields 146 pmol 32P - mg-1 protein. Without ouabain (20 degrees C, pH 7.0) less than 25% of the incorporation observed in the presence of ouabain is reached. Preincubation of the native microsomes with Mg2+ and K+, in order to decompose possibly present high-energy phosphoryl-bonds prior to ouabain treatment, does not affect the maximal phosphate incorporation. This indicates that the inorganic phosphate incorporation is not due to an exchange with high-energy phosphoryl-bonds, which might have been preserved in the microsomal preparations. Phosphorylation of the native microsomes by ATP in the presence of Mg2+ and Na+ reaches 90 and 50% maximal levels within 15--30 s at 0 degrees C and pH 7.4 at concentrations of [gamma-32P]ATP of 5--32 and 0.5--3.5 muM, respectively. The maximal phosphorylation level is 149 pmol 32P-mg-1 protein, equal to that of ouabain-treated microsomes phosphorylated by inorganic phosphate. Both inorganic phosphate and ATP phosphorylate on site per active enzyme subunit of 135 000 molecular weight. From the equilibrium constants for the phosphorylation of ouabain-treated microsomes by inorganic phosphate at 0 degrees C and 20 degrees C standard free-energy changes of --5.4 and --6.8 kcal/mol, respectively, are calculated. These values yield a standard enthalpy change of 14 kcal/mol and an entropy change of 70 cal/mol - degree K. This characterizes the reaction as a process driven by an entropy change. The intermediate formed by phosphorylation with Pi has maximal stability at acidic pH, as is the case for the intermediate formed with ATP. Solubilization in sodium dodecyl sulfate stabilizes the phosphoryl-bond in the pH range of 4--7. The non-solubilized preparation has optimal stability at pH 2--4, the level of which is equal to that of detergent-solubilized intermediate. Sodium dodecyl sulfate gel electrophoresis of the microsomes at pH 3, following incorporation of 32Pi yields 11 protein bands, only one of which (mol. wt 100 000--106 000) carries the radioactive label. This protein has the same molecular weight as the protein, which is phosphorylated by ATP in the presence of Mg2+ and Na+.     

Effect of magnesium ions on the thermostability of inorganic pyrophosphatase from baker's yeast]

The interaction of magnesium ions with inorganic pyrophosphatase from baker's yeast was studied by means of heat denaturation. The heat inactivation of this enzyme is a biphasic process. The velocities in the initial range and in the subsequent slower part of inactivation are diminished with rising Mg2+ concentration in the inactivation assay. A model is proposed which describes this behavior. It is assumed that two enzyme conformations exist in equilibrium whose conversion rates correspond to the inactivation rate in its order of magnitude. The equilibrium is shifted by Mg2+. The two enzyme species differ in their Mg2+ binding behavior as evidenced by differences in the half-saturation constants and the cooperativity of the binding. The same conclusions are drawn from the fluorimetric measurement of denaturation of inorganic pyrophosphatase. Besides, an additional Mg2+ binding site is demonstrable, the saturation of which obviously leads to stabilisation of part of the enzyme structure without protecting it against loss of enzymatic activity. With the same method the labilizing effect of Zn2+ on the structure of the inorganic pyrophosphatase from baker's yeast was studied.     

Divalent metal ion, inorganic phosphate, and inorganic phosphate analogue binding to yeast inorganic pyrophosphatase

Four different techniques, equilibrium dialysis, protection of enzymatic activity against chemical inactivation, 31P relaxation rats, and water proton relaxation rates, are used to study divalent metal ion, inorganic phosphate, and inorganic phosphate analogue binding to yeast inorganic pyrophosphatase, EC 3.6.1.1. A major new finding is that the binding of a third divalent metal ion per subunit, which has elsewhere been implicated as being necessary for enzymatic activity [Springs, B., Welsh, K. M., & Cooperman, B. S. (1981) Biochemistry (in press)], only becomes evident in the presence of added inorganic phosphate and that, reciprocally, inorganic phosphate binding to both its high- and low-affinity sites on the enzyme is markedly enhanced in the presence of divalent metal ions, with Mn2+ causing an especially large increase in affinity. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide evidence against divalent metal ion inner sphere binding to phosphate for enzyme subunits having one or two divalent metal ions bound per subunit and evidence for a conformational change restricting active-site accessibility to solvent on the binding of a third divalent metal ion per subunit.     

Inhibition of (Na+,K+)-ATPase by magnesium ions and inorganic phosphate and release of these ligands in the cycles of ATP hydrolysis

The kinetic data of magnesium and inorganic phosphate inhibition of the (Na+,K+)-dependent ATP hydrolysis are consistent with a model where both ligands act independently and their release in the ATPase cycle is an ordered process where inorganic phosphate is released first. The effects of magnesium on the stimulation of the ATPase activity by Na+, K+ and ATP, and the inhibition of that activity by inorganic phosphate, are consistent with Mg2+ acting both as a 'product' and as a dead-end inhibitor. The dead-end Mg-enzyme complex would be produced with an enzyme form located downstream in the reaction sequence from the point where Mg2+ acts as a 'product' inhibitor. In the absence of K+, Mg2+ inhibition was reduced when either Na+ or ATP concentrations were increased well beyond those concentrations needed to saturate their high-affinity sites. This ATP effect suggests that the dead-end Mg-enzyme complex formation is affected by the speed of the E2-E1 conformational change. The present model is consistent with the formation of an Mg-phosphoenzyme complex insensitive to K+ which could become K+-sensitive in the presence of high Na+ concentrations. These Mg-enzyme complexes appear as intermediaries in the Na+-ATPase activity found in the absence of external Na+ and K+. These results can be interpreted on the basis of Mg2+ binding to a single site in the enzyme molecule. In addition, these experiments provide kinetic evidence indicating that the stimulation by external Na+ of the ATPase activity in the absence of K+ is due to a K+-like action of Na+ on the external K+ sites.     

Heat capacity and entropy changes of the major isotype of the toad (Bufo) parvalbumin induced by calcium binding

The possible structural changes in the major isotype of parvalbumin from the toad (Bufo bufo japonicus) skeletal muscle caused by Ca2+ and Mg2+ binding have been analyzed by microcalorimetric titrations. Parvalbumin was titrated with Ca2+ in both the absence and presence of Mg2+ and with Mg2+ in the absence of Ca2+, at pH 7.0, and at 5 degrees, 15 degrees, and 25 degrees C. The two sites in a molecule were equivalent on Mg2(+)-Ca2+ exchange, but distinguishable on Ca2+ and Mg2+ binding. The reactions of parvalbumin with Ca2+ are exothermic at every temperature in both the absence and presence of Mg2+, but those with Mg2+ are always endothermic except for the binding to site 1 at 25 degrees C. The magnitudes of the hydrophobic and internal vibrational contributions to the heat capacity and entropy changes of parvalbumin on Ca2+ and Mg2+ binding and Mg2(+)-Ca2+ exchange have been estimated by the empirical method of Sturtevant [Sturtevant, J. M. (1977) Proc. Natl Acad. Sci. USA 74, 2236-2240]. Although no major conformational changes were noted between Ca2(+)- and Mg2(+)-bound forms of toad parvalbumin, the conformational difference was larger in Ca2+ (or Mg2+) binding to site 1 than site 2. This may indicate that the metal-free form is much less stable than any form with Ca2+ (or Mg2+) bound at one site at least. On Mg2(+)-Ca2+ exchange, the vibrational as well as hydrophobic entropy is only slightly increased in a parallel manner. In contrast, on Ca2+ (or Mg2+) binding, the hydrophobic entropy increases but the vibrational entropy decreases; the former indicates the sequestering of nonpolar groups from the surface to the interior of a molecule, and the latter suggests that the overall structures are tightened on Ca2+ (or Mg2+) binding but loosened on Mg2(+)-Ca2+ exchange. Despite the clear distinctions in the thermodynamic features, the conformational changes of toad parvalbumin are essentially the same as those of the two isotypes of bullfrog parvalbumins on Ca2+ binding and Mg2(+)-Ca2+ exchange.     

A functionally important Tyr-89 residue in Saccharomyces cerevisiae pyrophosphatase. II. A possible role in the mechanism of enzyme action]

Earlier it has been demonstrated that inactivation of inorganic pyrophosphatase (PPase) of S. cerevisiae by 7-chloro-4-nitronbenzofurasane is due to modification of Tyr89. The effect of pH and active center ligands on this reaction has been studied. It was found that pK for Tyr89 does not exceed 8.5; the phosphate-metal complex binding to the high affinity center protects Tyr89 from inactivation. Activating ions (Mg2+ and Zn2+) do not influence the inactivation, whereas the PPase inhibitor, Ca2+, enhances this process after saturation of the high affinity binding site. Saturation of two binding sites with Ca2+ has a protective effect on the enzyme. An increase in the rate of Tyr89 binding to the inhibitor in the presence of low concentrations of Ca2+ is due to the decrease of Tyr89 pK. The data obtained suggest that Tyr89 is located near the high affinity binding site for phosphate. The high reactivity of Tyr89 and its tight binding in the active center point to the presence of a hydrogen bondage with the substrate and suggest a role of a proton donor whose acceptor is the product of the enzymatic reaction, i.e., phosphate.     

Allosteric regulation of yeast inorganic pyrophosphatase by substrate

Kinetic and binding studies of yeast inorganic pyrophosphatase (EC 3.6.1.1) revealed a regulatory PPi-binding site. Rate vs substrate concentration dependencies were markedly nonhyperbolic in the range of 0.1-150 microM MgPPi at fixed Mg2+ levels of 0.05-10 mM provided that the enzyme had been preequilibrated with Mg2+. Imidodiphosphate, hydroxymethylenebisphosphonate, and phosphate eliminated the deviations from the Michaelis-Menten kinetics and inhibited PPi hydrolysis in a manner consistent with their binding at both active and regulatory sites. The results agreed with a model in which binding of uncomplexed PPi at the regulatory site markedly increases enzyme affinity for the activating Mg2+ ion. Ultrafiltration studies revealed the binding of at least 3 mol of the inhibitory hydroxymethylenebisphosphonate and of 2 mol of noninhibitory methylenebisphosphonate per mole of the dimeric enzyme.     

ADP, chloride ion, and metal ion binding to bovine brain glutamine synthetase

The binding of divalent cations and nucleotide to bovine brain glutamine synthetase and their effects on the activity of the enzyme were investigated. In ADP-supported gamma-glutamyl transfer at pH 7.2, kinetic analyses of saturation functions gave [S]0.5 values of approximately 1 microM for Mn2+, approximately 2 mM for Mg2+, 19 nM for ADP.Mn, and 7.2 microM for ADP.Mg. The method of continuous variation applied to the Mn2+-supported reaction indicated that all subunits of the purified enzyme express activity when 1.0 equiv of ADP is bound per subunit. Measurements of equilibrium binding of Mn2+ to the enzyme in the absence and presence of ADP were consistent with each subunit binding free Mn2+ (KA approximately equal to 1.5 X 10(5) M-1) before binding the Mn.ADP complex (KA' approximately equal to 1.1 X 10(6) M-1). The binding of the first Mn2+ or Mg2+ to each subunit produces structural perturbations in the octameric enzyme, as evidenced by UV spectral and tryptophanyl residue fluorescence changes. The enzyme, therefore, has one structural site per subunit for Mn2+ or Mg2+ and a second site per subunit for the metal ion-nucleotide complex, both of which must be filled for activity expression. Chloride binding (KA' approximately equal to 10(4) M-1) to the enzyme was found to have a specific effect on the protein conformation, producing a substantial (30%) quench of tryptophanyl fluorescence and increasing the affinity of the enzyme 2-4-fold for Mg2+ or Mn2+. Arsenate, which activates the gamma-glutamyl transfer activity by binding to an allosteric site, and L-glutamate also cause conformational changes similar to those produced by Cl- binding. Anion binding to allosteric sites and divalent metal ion binding at active sites both produce tryptophanyl residue exposure and tyrosyl residue burial without changing the quaternary enzyme structure.     

Phosphorylation of calcium adenosinetriphosphatase by inorganic phosphate: reversible inhibition at high magnesium ion concentrations

Magnesium stimulates phosphorylation of the calcium pump protein of the sarcoplasmic reticulum by inorganic phosphate, but the effect is reversed by high [Mg2+]. This reversal is readily explained in terms of the generally accepted existence of two conformational states of the enzyme, E1 and E2. E2 is the form of the enzyme that can be phosphorylated by Pi, and it has one binding site for Mg2+. E1 is the form of the enzyme that has two high-affinity Ca2+ binding sites, and it is phosphorylated by ATP when Ca2+ is bound. Mg2+ can bind weakly to the two Ca2+ sites and to a third site known to be present on E1; this stabilizes E1 at the expense of E2 when [Mg2+] is large. Stabilization of E1 at pH 6.2 and 25 degrees C was found to be a highly cooperative function of [Mg2+] and was not prevented by increasing [Pi]. The latter result requires the existence of a binding site for Pi on E1, with an affinity for Pi comparable to that of E2. Cooperativity with respect to [Mg2+] requires that E2 is the stable state of the enzyme in the absence of ligands, with an equilibrium constant [E2]/[E1] on the order of 10(3) or higher at pH 6.2 and 25 degrees C.     

Kinetics and thermodynamics of catalysis by the inorganic pyrophosphatase of Escherichia coli in both directions

Combined evidence obtained from the measurements of pyrophosphate hydrolysis and synthesis, oxygen exchange between phosphate and water, enzyme-bound pyrophosphate formation and Mg2+ binding enabled us to deduce the overall scheme of catalysis by Escherichia coli inorganic pyrophosphatase in the presence of Mg2+. We determined the equilibrium constants for Mg2+ binding to various enzyme species and forward and reverse rate constants for the four steps of the catalytic reaction, namely, binding/release of PPi, hydrolysis/synthesis of PPi and successive binding/release of two Pi molecules. Catalysis by the E. coli enzyme in both directions, in contrast to baker's yeast pyrophosphatase, occurs via a single pathway, which requires the binding of Mg2+ to the sites of four types. Three of them can be filled in the absence of the substrates, and the affinity of one of them to Mg2+ is increased by two orders of magnitude in the enzyme-substrate complexes. The distribution of 18O-labelled phosphate isotopomers during the exchange indicated that hydrolysis of pyrophosphate in the active site is appreciably reversible. The equilibrium constant for this process estimated from direct measurements is 5.0. The ratio of the maximal velocities of pyrophosphate hydrolysis and synthesis is 69. The rate of the synthesis is almost entirely determined by the rate of the release of pyrophosphate from the enzyme. In the hydrolytic reaction, enzyme-bound pyrophosphate hydrolysis and successive release of two phosphate molecules proceed with nearly equal rate constants.     

Tyrosine-89 is important for enzymatic activity of S. cerevisiae inorganic pyrophosphatase.

7-Chloro-4-nitro-benzofurazan selectively modifies one PPase Tyr residue per subunit and lowers the enzyme activity. Hydrolysis of the modified protein by trypsin and then by chymotrypsin produces the 82-89 peptide which possesses modified Tyr-89. Substrate analog (CaPPi) and the product of the enzyme reaction, MgPi, protect the enzyme against inactivation. Ions of metal-activators (Mg2+, Zn2+) exert no influence on the inactivation rate. On the contrary, the Ca(2+)-inhibitor of the enzyme accelerates the reaction by binding to the high-affinity site, and effectively decreases it when Ca2+ binds to both sites. Mg2+ competes with Ca2+ for one binding site, which is the low affinity site for Mg2+ and the high-affinity site for Ca2+. The Ca2+ saturation of the high-affinity site decreases the pK2 of Tyr-89, probably due to direct coordination between Tyr and Ca2+. The observed properties of Tyr-89 modification enable us to propose that Tyr-89 serves as a proton donor for phosphate releasing during enzymatic hydrolysis of pyrophosphate. The Ca2+ inhibitory effect on the enzyme activity may be due to the existence of a Tyr-89 bond in the Ca2+ pyrophosphatase complex.     

Binding of two Sr2+ ions changes the chemical specificities for phosphorylation of the sarcoplasmic reticulum calcium ATPase through a stepwise mechanism.

The sequential binding of Sr2+ and Ca2+ to the cytoplasmic transport sites of the sarcoplasmic reticulum calcium ATPase allows the formation of two different mixed complexes: cE.Sr.Ca, with Sr2+ bound to the "inner" site and Ca2+ bound to the "outer" site, and cE. Ca.Sr, with Ca2+ bound to the inner site and Sr2+ bound to the outer site (pH 7.0, 25 degrees C, 10 mM MgCl2, 100 mM KCl). Both cE.Sr.45Ca and cE.45Ca.Sr react with ATP to internalize one 45Ca/phosphoenzyme. The value of K0.5 = 83 microM Sr2+ for activation of the enzyme for phosphorylation by ATP is much larger than K0.5 = 28 microM Sr2+ for inhibition of phosphoenzyme formation from inorganic phosphate (eta H = 1.0-1.3). These results are consistent with the sequential binding of two strontium ions with negative cooperativity and dissociation constants of KSr1 = 35 microM and KSr2 = 55 microM. The species cE.Sr2 and cE.Ca2 react rapidly with ATP but not inorganic phosphate. However, enzyme with one strontium bound, cE.Sr, does not react with either inorganic phosphate or ATP. Therefore, the conformational changes in the enzyme that alter the chemical specificity for phosphorylation by ATP and by inorganic phosphate are different. This requires the existence of at least three forms of the unphosphorylated enzyme with three different chemical specificities for catalysis.     

Effects of trifluoperazine on calcium binding by calmodulin. Heat capacity and entropy changes

The possible structural changes of the calmodulin-trifluoperazine (TFP) complex caused by Ca2+ binding have been analyzed by microcalorimetric titrations. Titrations of calmodulin with Ca2+ in the presence of 8-fold molar excess TFP have been made both in the absence and presence of Mg2+, at pH 7.0, and at 5, 15, and 25 degrees C. At high concentrations of TFP calmodulin forms a complex with TFP even in the absence of Ca2+. The reaction of the calmodulin-TFP complex with Ca2+ is exothermic, both in the presence and absence of Mg2+. In the presence of Mg2+ the reaction is driven almost entirely by a favorable enthalpy change. The magnitudes of the hydrophobic and internal vibrational contributions to the heat capacity and entropy changes of this complex on Ca2+ binding have been estimated by the empirical method of Sturtevant (Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 2236-2240). In the presence of Mg2+, the vibrational as well as hydrophobic entropy is slightly increased in a parallel manner by Ca2+ binding to each of the binding sites. In contrast, when Mg2+ is absent, the hydrophobic entropy gradually increases on Ca2+ binding, but the vibrational entropy decreases. These changes of entropy indicate the assembling of non-polar groups on the surface of the complex and suggest that the overall structure is loosened in the presence of Mg2+, but tightened in the absence of Mg2+.     

Equilibrium binding of coenzymes and substrates to nicotinamide-adenine dinucleotide phosphate-linked isocitrate dehydrogenase from bovine heart mitochondria.

1. The stoicheiometries and affinities of ligand binding to isocitrate dehydrogenase were studied at pH 7.0, mainly by measuring changes in NADPH and protein fluorescence. 2. The affinity of the enzyme for NADPH is about 100-fold greater than it is for NADP+ in various buffer/salt solutions, and the affinities for both coenzymes are decreased by Mg2+, phosphate and increase in ionic strength. 3. The maximum binding capacity of the dimeric enzyme for NADPH, from coenzyme fluorescence and protein-fluorescence measurements, and also for NADP+, by ultrafiltration, is 2 mol/mol of enzyme. Protein-fluorescence titrations of the enzyme with NADP+ are apparently inconsistent with this conclusion, indicating that the increase in protein fluorescence caused by NADP+ binding is not proportional to fractional saturation of the binding sites. 4. Changes in protein fluorescence caused by changes in ionic strength and by the binding of substrates, Mg2+ or NADP+ (but not NADPH) are relatively slow, suggesting conformation changes. 5. In the presence of Mg2+, the enzyme binds isocitrate very strongly, and 2-oxoglutarate rather weakly. 6. Evidence is presented for the formation of an abortive complex of enzyme-Mg2+-isocitrate-NADPH in which isocitrate and NADPH are bound much more weakly than in their complexes with enzyme and Mg2+ alone. 7. The results are discussed in relation to the interpretation of the kinetic properties of the enzyme and its behaviour in the mitochondrion.     

Enthalpy, entropy and heat capacity changes induced by binding of calcium ions to cardiac troponin C

Microcalorimetric titrations have been used to study the binding of Ca2+ to cardiac troponin C. Measurements were made both in the presence and in the absence of Mg2+, and at temperatures of 5 degrees, 15 degrees and 25 degrees C. Changes in enthalpy, entropy and heat capacity of troponin C associated with Ca binding have been determined. Cardiac troponin C exhibited a decrease in enthalpy and an increase in entropy associated with Ca binding. Enthalpy changes increased linearly with temperature, indicating that the Ca binding causes negative changes in the heat capacity of troponin C. These results show that the Ca binding causes a strong hydrophobic effect and a tightening of the molecular structure of cardiac troponin C.     

Thermodynamics, kinetics, and mechanism in yeast inorganic pyrophosphatase catalysis of inorganic pyrophosphate: inorganic phosphate equilibration

We have developed two methods for quantitatively measuring inorganic pyrophosphate (PPi) in the presence of 10(3)--10(4) molar excesses of inorganic phosphate (Pi) and used them to measure the extent of enzyme-bound pyrophosphate (EPPi) formation in solutions of yeast inorganic pyrophosphatase and Pi. We have also measured the rate of enzyme-catalyzed H2O--phosphate oxygen exchange. We find both processes to have essentially identical dependence on Mg2+ and Pi concentrations, thus providing important confirmation for the recent proposal by Janson et al. (1979) that oxygen exchange proceeds via EPPi formation. Our results are consistent with a model in which three Mg2+ per active site are required for EPPi formation but inconsistent with a model requiring only two Mg2+ per active site and permit the formulation of an overall scheme for inorganic pyrophosphatase catalysis of PPi--Pi equilibration as well as the evaluation of equilibrium and rate constants in this scheme. The major results and conclusions of our work are the following: (a) the equilibrium constant for PPi (enzyme-bound) in equilibrium with 2Pi (enzyme-bound) is 4.8; (b) following PPi hydrolysis, the first released Pi contains an oxygen from solvent water; (c) the steps for PPi hydrolysis on the enzyme and for release of both product Pi's are all partially rate determining in overall enzyme-catalyzed PPi hydrolysis; (d) PPi formation on the enzyme is rate determining for H2O--Pi oxygen exchange; (e) PPi dissociation from the enzyme is very slow and is the rate-determining step in Pi--PPi exchange (Cohn, 1958; Janson et al., 1979). This also accounts for the observation that the calculated dissociation constant for MgPPi complex binding to enzyme is considerably lower than the measured Km for enzyme-catalyzed MgPPi hydrolysis.     

Modification of ligand binding to the Na+/K+-activated ATPase

Interactions between the ligands Mg2+, K+, and substrate and the Na+/K+-activated ATPase were examined in terms of a rapid-equilibrium, random-order, terreactant kinetic scheme for the K+-nitrophenyl phosphatase reaction that is catalyzed by this enzyme. At 37 degrees C and pH 7.5 the derived values for the dissociation constants from the free enzyme were 0.2, 0.08, and 1.4 mM for Mg2+, K+, and substrate, respectively. For Mg2+ interactions, the presence of 20% (v/v) dimethyl sulfoxide (Me2SO) increased the calculated affinity 25-fold; higher concentrations increased affinity still further. Neither reducing the temperature to 20 degrees C nor altering the pH from 6.5 to 8.3 appreciably changed the affinity for Mg2+ in the absence or presence of Me2SO. The Mg2+ sites are thus characterized by an absence of functional groups ionizable in the pH range 6.5-8.3, with binding driven by entropy changes, and with Me2SO, probably through solvation effects on the protein, increasing affinity for Mg2+ close to that for Ca2+ and Mn2+. By contrast, for K+ interactions, the presence of 20% Me2SO increased the calculated affinity only by half; moreover, reducing the temperature to 20 degrees C and the pH to 6.5 both increased affinity and diminished the response to Me2SO. The K+ sites are thus characterized by a marked sensitivity to pH and temperature, presumably through alterations in enzyme conformational equilibria that in turn are modifiable by Me2SO. Inhibition by higher concentrations of Mg2+, which varies inversely with the K+ concentration, was decreased by Me2SO. Finally, for substrate interactions, the presence of 20% Me2SO increased the calculated affinity 4-fold, and, as for Mg2+-binding, neither reducing the temperature nor varying the pH over the range 6.5-8.3 appreciably altered the affinity in the absence or presence of Me2SO. Thus, the substrate sites, like the Mg2+ sites, are characterized by an absence of functional groups ionizable in this range, with binding driven by entropy changes, and with Me2SO increasing affinity for substrate, in this case probably through favoring the partitioning of substrate from the medium into the hydrophobic active site.