Monovalent Cation Site Research Articles

Chemical stabilization of Eu2+ in LuPO4 and YPO4 hosts and its peculiar sharp line luminescence

The efficient 4f65d→4f7 (d-f) luminescence from Eu2+ is well-known for host lattices where Eu2+ is substituted on a divalent (e.g. Ca2+, Sr2+) or monovalent (e.g. Na+, K+) cation site. Only recently some studies appeared concerning d-f emission from Eu2+ on a trivalent cation site. The stable Eu2+ emission was observed in X-ray irradiated LuPO4:Eu3+ crystals where Eu2+ is produced through trapping of conduction band electrons generated by X-ray absorption. Here, we demonstrated that Eu2+ can also be chemically stabilized in YPO4 and LuPO4 through co-doping with a tetravalent charge compensator: Hf4+. In orthophosphate crystals doubly doped with Eu and Hf, the characteristic Eu2+ emission is observed at low temperatures without the need for X-ray irradiation. The excitation and emission spectra show strong and narrow zero-phonon lines and a rich vibronic structure. In the excitation spectra a multitude of zero-phonon transitions to individual crystal field components of 7FJ multiplets in the 4f6(7FJ)5d excited state was observed for Eu2+ in both LuPO4 and YPO4. Analysis of the spectra is consistent with a slightly larger crystal field splitting for Eu2+ on the smaller Lu3+ site (compared to Y3+). The observation of many 4f6(7FJ)5d electronic origins make these systems to be model compounds for testing the validity of theoretical models for energy level calculations of 4f65d states of Eu2+.

Structural basis of elongation factor 2 switching.

Archaebacterial and eukaryotic elongation factor 2 (EF-2) and bacterial elongation factor G (EF-G) are five domain GTPases that catalyze the ribosomal translocation of tRNA and mRNA. In the classical mechanism of activation, GTPases are switched on through GDP/GTP exchange, which is accompanied by the ordering of two flexible segments called switch I and II. However, crystal structures of EF-2 and EF-G have thus far not revealed the conformations required by the classical mechanism. Here, we describe crystal structures of Methanoperedens nitroreducens EF-2 (MnEF-2) and MnEF-2-H595N bound to GMPPCP (GppCp) and magnesium displaying previously unreported compact conformations. Domain III forms interfaces with the other four domains and the overall conformations resemble that of SNU114, the eukaryotic spliceosomal GTPase. The gamma phosphate of GMPPCP is detected through interactions with switch I and a P-loop structural element. Switch II is highly ordered whereas switch I shows a variable degree of ordering. The ordered state results in a tight interdomain arrangement of domains I-III and the formation of a portion of a predicted monovalent cation site involving the P-loop and switch I. The side chain of an essential histidine residue in switch II is placed in the inactive conformation observed for the “on” state of elongation factor EF-Tu. The compact conformations of MnEF-2 and MnEF-2-H595N suggest an “on” ribosome-free conformational state.

Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme.

Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate-lyase. We show that PFL-AE binds a catalytically essential monovalent cation at its active site, yet another parallel with B12 enzymes, and we characterize this cation site by a combination of structural, biochemical, and spectroscopic approaches. Refinement of the PFL-AE crystal structure reveals Na+ as the most likely ion present in the solved structures, and pulsed electron nuclear double resonance (ENDOR) demonstrates that the same cation site is occupied by 23Na in the solution state of the as-isolated enzyme. A SAM carboxylate-oxygen is an M+ ligand, and EPR and circular dichroism spectroscopies reveal that both the site occupancy and the identity of the cation perturb the electronic properties of the SAM-chelated iron–sulfur cluster. ENDOR studies of the PFL-AE/[13C-methyl]-SAM complex show that the target sulfonium positioning varies with the cation, while the observation of an isotropic hyperfine coupling to the cation by ENDOR measurements establishes its intimate, SAM-mediated interaction with the cluster. This monovalent cation site controls enzyme activity: (i) PFL-AE in the absence of any simple monovalent cations has little–no activity; and (ii) among monocations, going down Group 1 of the periodic table from Li+ to Cs+, PFL-AE activity sharply maximizes at K+, with NH4+ closely matching the efficacy of K+. PFL-AE is thus a type I M+-activated enzyme whose M+ controls reactivity by interactions with the cosubstrate, SAM, which is bound to the catalytic iron–sulfur cluster.

Tryptophan Synthase: Structure and Function of the Monovalent Cation Site

The monovalent cation (MVC) site of the tryptophan synthase from Salmonella typhimurium plays essential roles in catalysis and in the regulation of substrate channeling. In vitro, MVCs affect the equilibrium distribution of intermediates formed in the reaction of l-Ser with the alpha(2)beta(2) complex; the MVC-free, Cs(+)-bound, and NH(4)(+)-bound enzymes stabilize the alpha-aminoacrylate species, E(A-A), while Na(+) binding stabilizes the l-Ser external aldimine species, E(Aex(1)). Two probes of beta-site reactivity and conformation were used herein, the reactive indole analogue, indoline, and the l-Trp analogue, l-His. MVC-bound E(A-A) reacts rapidly with indoline to give the indoline quinonoid species, E(Q)(indoline), which slowly converts to dihydroiso-l-tryptophan. MVC-free E(A-A) gives very little E(Q)(indoline), and turnover is strongly impaired; the fraction of E(Q)(indoline) formed is <3.5% of that given by the Na(+)-bound form. The reaction of l-Ser with the MVC-free internal aldimine species, E(Ain), initially gives small amounts of an active E(A-A) which converts to an inactive species on a slower, conformational, time scale. This inactivation is abolished by the binding of MVCs. The inactive E(A-A) appears to have a closed beta-subunit conformation with an altered substrate binding site that is different from the known conformations of tryptophan synthase. Reaction of l-His with E(Ain) gives an equilibrating mixture of external aldimine and quinonoid species, E(Aex)(his) and E(Q)(his). The MVC-free and Na(+) forms of the enzyme gave trace amounts of E(Q)(his) ( approximately 1% of the beta-sites). The Cs(+) and NH(4)(+) forms gave approximately 17 and approximately 14%, respectively. The reactivity of MVC-free E(Ain) was restored by the binding of an alpha-site ligand. These studies show MVCs and alpha-site ligands act synergistically to modulate the switching of the beta-subunit from the open to the closed conformation, and this switching is crucial to the regulation of beta-site catalytic activity. Comparison of the structures of Na(+) and Cs(+) forms of the enzyme shows Cs(+) favors complexes with open indole binding sites poised for the conformational transition to the closed state, whereas the Na(+) form does not. The beta-subunits of Cs(+) complexes exhibit preformed indole subsites; the indole subsites of the open Na(+) complexes are collapsed, distorted, and too small to accommodate indole.

NEUTRON SPECTROSCOPIC STUDY OF SYNTHETIC ALUNITE AND OXONIUM-SUBSTITUTED ALUNITE

Synthetic, polycrystalline samples of alunite [K 0.88 (H 3 O) 0.12 Al 2.64 (SO 4 ) 2 (OH) 4.92 (H 2 O) 1.08 ] and oxonium-substituted alunite [H 3 OAl 2.87 (SO 4 ) 2 (OH) 5.61 (H 2 O) 0.39 ] have been investigated using incoherent, inelastic neutron-scattering (IINS) methods in order to determine the nature of the non-OH “H 2 O”. IINS measurements were made on non-deuterated samples at 20 K using the HRMECS chopper spectrometer with 250 and 600 meV incident energies. Alunite and oxonium-substituted alunite exhibit similar spectral features over the energy range of 120–550 meV, where assignments for local vibrations can be made based on the presence of OH and H 2 O groups. Salient differences in the intensities and positions of the observed low-energy vibrational bands reflect the effects of chemical substitution on the structural environment of the monovalent cation site and neighboring Al and OH sites in the framework.

A novel deamido-NAD+-binding site revealed by the trapped NAD-adenylate intermediate in the NAD+ synthetase structure.

Nicotinamide adenine dinucleotide (NAD+) has a central role in life processes. The ubiquitous enzyme NAD+ synthetase catalyzes a key step in NAD+ biosynthesis, transforming deamido-NAD+ into NAD+ by a two-step reaction. NAD+ synthetase belongs to the amidotransferase family and has been recognized as a member of the family of N-type ATP pyrophosphatases. In order to investigate the mechanism of the reaction carried out by NAD+ synthetase we have determined a high-resolution three-dimensional structure of the Bacillus subtilis homodimeric NAD+ synthetase in complex with the trapped reaction intermediate NAD-adenylate. Two NAD-adenylate molecules and two pyrophosphate (PPi) molecules are observed in the 1.3 A resolution structure of the NAD+ synthetase-NAD-adenylate complex. Structural studies on the NAD+ synthetase-NAD-adenylate adduct and on the cation-binding sites reveal a new deamido-NAD+-binding site located at the subunit interface, locate a binuclear magnesium cluster at the ATP-binding site and, identify two monovalent cation sites, one of which may represent an ammonium-binding site. Our results suggest that two different catalytic strategies have been adopted by NAD+ synthetase in the two different steps of the reaction. During the adenylation step, no protein residues seem to be located properly to directly participate in catalysis, which is likely to be carried out with the fundamental assistance of an electron-withdrawing trimetallic constellation present in the active site. A different behavior is observed for the second step, in which an ammonium ion is the binding species. In this step, Asp173 is a key residue in both deprotonation of the primarily bound ammonium ion, and stabilization of the tetrahedral transition-state intermediate. Moreover, the structural data suggest that product release can take place only after all substrates are bound to the enzyme, and product release is ultimately controlled by the conformation adopted by two mobile loops.

From sequence analysis of three novel ascorbate peroxidases from Arabidopsis thaliana to structure, function and evolution of seven types of ascorbate peroxidase.

Ascorbate peroxidases are haem proteins that efficiently scavenge H2O2 in the cytosol and chloroplasts of plants. Database analyses retrieved 52 expressed sequence tags coding for Arabidopsis thaliana ascorbate peroxidases. Complete sequencing of non-redundant clones revealed three novel types in addition to the two cytosol types described previously in Arabidopsis. Analysis of sequence data available for all plant ascorbate peroxidases resulted in the following classification: two types of cytosol soluble ascorbate peroxidase designated cs1 and cs2; three types of cytosol membrane-bound ascorbate peroxidase, namely cm1, bound to microbodies via a C-terminal membrane-spanning segment, and cm2 and cm3, both of unknown location; two types of chloroplast ascorbate peroxidase with N-terminal transit sequences, the stromal ascorbate peroxidase (chs), and the thylakoid-bound ascorbate peroxidase showing a C-terminal transmembrane segment and designated cht. Further comparison of the patterns of conserved residues and the crystal structure of pea ascorbate peroxidase showed that active site residues are conserved, and three peptide segments implicated in interaction with reducing substrate are similar, excepting cm2 and cm3 types. A change of Phe-175 in cytosol types to Trp-175 in chloroplast types might explain the greater ascorbate specificity of chloroplast compared with cytosol ascorbate peroxidases. Residues involved in homodimeric subunit interaction are conserved only in cs1, cs2 and cm1 types. The proximal cation (K+)-binding site observed in pea ascorbate peroxidase seems to be conserved. In addition, cm1, cm2, cm3, chs and cht ascorbate peroxidases contain Asp-43, Asn-57 and Ser-59, indicative of a distal monovalent cation site. The data support the hypothesis that present-day peroxidases evolved by an early gene duplication event.

Cation-Binding Location and Hydrogen-Exchange Sites for Gramicidin in SDS Micelles Using NOESY NMR

The site of monovalent cation binding and sites of hydrogen exchange between amide protons and water molecules in the gramicidin A and Phe-1 gramicidin A channels incorporated into SDS micelles have been determined using a NOESY NMR technique. The cation-binding pocket was found to involve residues 10–15 of the peptide.

Discovery of the ammonium substrate site on glutamine synthetase, A third cation binding site

Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and glutamate to yield glutamine, ADP, and inorganic phosphate in the presence of divalent cations. Bacterial GS is an enzyme of 12 identical subunits, arranged in two rings of 6, with the active site between each pair of subunits in a ring. In earlier work, we have reported the locations within the funnel-shaped active site of the substrates glutamate and ATP and of the two divalent cations, but the site for ammonia (or ammonium) has remained elusive. Here we report the discovery by X-ray crystallography of a binding site on GS for monovalent cations, Tl+ and Cs+, which is probably the binding site for the substrate ammonium ion. Fourier difference maps show the following. (1) Tl+ and Cs+ bind at essentially the same site, with ligands being Glu 212, Tyr 179, Asp 50', Ser 53' of the adjacent subunit, and the substrate glutamate. From its position adjacent to the substrate glutamate and the cofactor ADP, we propose that this monovalent cation site is the substrate ammonium ion binding site. This proposal is supported by enzyme kinetics. Our kinetic measurements show that Tl+, Cs+, and NH4+ are competitive inhibitors to NH2OH in the gamma-glutamyl transfer reaction. (2) GS is a trimetallic enzyme containing two divalent cation sites (n1, n2) and one monovalent cation site per subunit. These three closely spaced ions are all at the active site: the distance between n1 and n2 is 6 A, between n1 and Tl+ is 4 A, and between n2 and Tl+ is 7 A. Glu 212 and the substrate glutamate are bridging ligands for the n1 ion and Tl+. (3) The presence of a monovalent cation in this site may enhance the structural stability of GS, because of its effect of balancing the negative charges of the substrate glutamate and its ligands and because of strengthening the "side-to-side" intersubunit interaction through the cation-protein bonding. (4) The presence of the cofactor ADP increases the Tl+ binding to GS because ADP binding induces movement of Asp 50' toward this monovalent cation site, essentially forming the site. This observation supports a two-step mechanism with ordered substrate binding: ATP first binds to GS, then Glu binds and attacks ATP to form gamma-glutamyl phosphate and ADP, which complete the ammonium binding site. The third substrate, an ammonium ion, then binds to GS, and then loses a proton to form the more active species ammonia, which attacks the gamma-glutamyl phosphate to yield Gln. (5) Because the products (Glu or Gln) of the reactions catalyzed by GS are determined by the molecule (water or ammonium) attacking the intermediate gamma-glutamyl phosphate, this negatively charged ammonium binding pocket has been designed naturally for high affinity of ammonium to GS, permitting glutamine synthesis to proceed in aqueous solution.

In squid axons the Ca i2+ regulatory site of the Na + /Ca 2+ exchanger is drastically modified by sulfhydryl blocking agents. Evidences that intracellular Ca i2+ regulatory and transport sites are different

We have explored the effect of the sulfhydryl group blocker p-chloromercuryphenylsulfonic acid (PCMBS) on Ca 2+ and Na + interactions with the Na − /Ca 2+ exchanger in squid giant nerve fibers. Steady-state Na o +-dependent Ca 2+ efflux (forward) and Na i +-dependent Ca 2+ influx (reverse) were measured in internally dialyzed, voltage clamped squid axons. External PCMBS (0.5 mM, for 25–35 min) has no effect on the activation of Ca 2+ efflux by Na o +, and Ca o 2+ or on the activatory external monovalent cation site. In contrast, when applied internally it drastically reduces the affinity of the Na + /Ca 2+ exchanger towards Ca i 2+ ions without affecting its maximal rate of transport; in the presence of MgATP the K 0.5 for Ca i 2+ activation of forward Na + /Ca 2+ exchange increases from 1.5 μM to 95 μM; likewise the apparent affinity of the Ca i 2+ stimulation of the reversal exchange decreases 100-fold. Interestingly, no effect of PCMBS was found on the interactions between Na i + and Ca i 2+ ions with the internal transport site(s) (inhibition of Na o + and Ca o 2+-dependent Ca 2+ efflux by Na i +). On the other hand, Na i + ions do not modify the interactions of Ca i 2+ with that site. Two important characteristics of the Ca i 2+ regulatory site are uncover in this work: (i) sulfhydryl groups are important in maintaining the integrity of the Ca 2+ binding domain of the Ca i 2+ regulatory site and (ii) Na i + and Ca i 2+ regulatory, or Na i + and Ca i 2+ transporting sites, are different entities.

Characterization of lanthanides as competitors of Na+ and K+ in occlusion sites of renal (Na+,K+)-ATPase

Lanthanides are useful probes in Ca2+ binding proteins, including sarcoplasmic reticulum (Ca2+,Mg2+)-ATPase. Here, we report that lanthanides compete with Rb+ and Na+ for occlusion in renal (Na+,K+)-ATPase. The lanthanides appear to bind at a single site and act as competitive antagonists, without themselves becoming occluded. All lanthanides tested are effective with the order of potencies Pr greater than Nd greater than La greater than Eu greater than Tb greater than Ho greater than Er, but differences are small. The presence of Mg2+ ions does not affect competition of La3+ with Na+ or K+ suggesting that the effects are not exerted via divalent metal sites. Lanthanides compete with Rb+ and Na+ in membranes digested with trypsin so as to produce 19-kDa and smaller fragments of the alpha-chain (Karlish, S.J.D., Goldshleger, R., and Stein, W. D. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4566-4570), also suggestive of a direct interaction of lanthanides with Na+ and K+ sites. Effects of lanthanides on conformational changes of fluorescein-labeled (Na+,K+)-ATPase are Na(+)-like. They stabilize the E1 state and compete with K+ ions. The Ki for La3+ is 0.445 microM. The apparent affinity in fluorescence assays is proportional to enzyme concentration (Ki = 32.4*[protein] + 0.445 microM La3+), suggesting that lanthanides are also bound nonspecifically (possibly to phospholipids). Direct assays confirm that Tb3+ binding is nonspecific. Measurements of the rate of various conformational transitions show that the rate of E2(K+)----E1(X) (X = Na+ or La3+) is significantly inhibited by La3+ compared to Na+. La3+ ions also slightly accelerate the rate of the E1----E2(K+) conformational transition. The dissociation rate of La3+ has been measured by monitoring the rate of E1(La3+)----E2(K+). It is 1.741 s-1 at 25 degrees C. Based on this value, it is unlikely that La3+ ions are stably occluded, consistent with the conclusion from occlusion experiments. In the future, lanthanides bound to monovalent cation sites with high affinity may become useful probes for location and characterization of sites, although it will be necessary to take into account the large amount of nonspecific binding.

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

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

Asymmetrical properties of the Na-Ca exchanger in voltage-clamped, internally dialyzed squid axons under symmetrical ionic conditions.

In this work we have investigated whether the asymmetrical properties of the Na/Ca exchange process found in intact preparations are intrinsic to the exchange protein(s) or the result of the asymmetric ionic environment normally prevailing in living cells. The activation of the Na/Ca exchanger by Ca2+ ions, monovalent cations, ATP gamma S and the effect of membrane potential on the different operational modes of the exchanger (Nao/Cai, Cao/Nai, Cao/Cai, and Nao/Nai) was studied in voltage-clamped squid giant axons externally perfused and internally dialyzed with symmetrical ionic solutions. Under these conditions: (a) Ca ions activate with higher affinity from the inside (K1/2 = 22 microM) than from the outside (K1/2 = 300 microM); (b) experiments measuring the Cao-dependent Ca efflux in the conditions Lio-Trisi, Lio-Lii, Triso-Trisi, and Triso-Lii, show that the activating monovalent cation site on the exchanger faces the external surface; (c) ATP gamma S activates the Cao-dependent Ca efflux (Cao/Cai exchange) only at nonsaturating [Ca2+]i. Its effect appears to be on the Ca transport site since no alteration in the apparent affinity of the activating monovalent cation site was observed. The above results show that the Na/Ca exchange process is indeed a highly asymmetric transport mechanism. Finally, the voltage dependence of the components of the different exchange modes was measured over the range of +20 to -40 mV. The voltage dependence (approximately 26% change/25 mV) was found to be similar for all modes of operation of the exchanger except Nao/Nai exchange, which was found to be voltage insensitive. The sensitivity of the Cao/Cai exchange to voltage was found to be the same in the presence and in the complete absence of monovalent cations. This finding does not support the proposition that the voltage sensitivity of the Cao/Cao exchange is induced by the binding and transport of an external monovalent cation.

Interaction of a K(+)-competitive inhibitor, a substituted imidazo[1,2a] pyridine, with the phospho- and dephosphoenzyme forms of H+, K(+)-ATPase.

Defining the structural and catalytic properties of the ion transport site(s) of enzyme-phosphorylating ATPases is of key importance in understanding the mechanism of ion transport by these enzymes. In the case of the H+, K(+)-ATPase, SCH 28080 (3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)imidazo[1,2a]-pyridine) has been shown to act as a high affinity, extracytosolic, K(+)-competitive inhibitor of Mg2+, K(+)-ATPase activity (Wallmark, B., Briving, C., Fryklund, J., Munson, K., Jackson, R., Mendlein, J., Rabon, E., and Sachs, G. (1987) J. Biol. Chem. 262, 2077-2084). To define the nature of the SCH 28080-binding site in relation to the catalytic cycle of the enzyme, we have investigated the effects of this potential K+ transport site probe on the steady-state and partial reactions of the H+, K(+)-ATPase. In the absence of K+, SCH 28080 inhibits Mg2(+)-ATPase activity with high affinity (apparent Ki = 30 nM). Inhibition is due to K(+)-like prevention of phosphoenzyme formation. SCH 28080 has no effect on Mg2(+)-catalyzed dephosphorylation. SCH 28080, at concentrations less than 0.5 microM, increases the apparent Km for K+ for Mg2+, K(+)-ATPase activity with little effect on the maximum velocity. At higher concentrations of SCH 28080, reversal of inhibition by higher K+ concentrations is not complete, due to inhibition of ATPase activity by high K+. In contrast, SCH 28080 inhibits K(+)-stimulated dephosphorylation by competitively displacing K+ from phosphoenzyme with an extracytosolic conformation of the monovalent cation site (E2P) at low concentrations of SCH 28080 and K+. At higher concentrations, 10 microM SCH 28080 and 50 mM K+, a slowly dephosphorylating complex with both SCH 28080 and K+ bound to E2P may form which represents a small fraction of the total E2P (15-25%). Preincubation of SCH 28080 with E2P completely blocks K(+)-stimulated dephosphorylation, and K+ is unable to reverse this preincubation effect, indicating that the SCH 28080 dissociation rate is at least as slow as K(+)-independent dephosphorylation of E2P. These findings indicate that SCH 28080 inhibits K(+)-stimulated ATPase activity by competing with K+ for binding to E2P and blocking K(+)-stimulated dephosphorylation. In the absence of K+, SCH 28080 has a higher apparent affinity for E2P, but it permits K(+)-independent dephosphorylation. Since the dissociation rate of SCH 28080 from the enzyme is slow, phosphoenzyme formation is prevented by SCH 28080 remaining bound to the extracytosolic conformation of the monovalent cation site, thereby reducing the steady-state level of phosphoenzyme.

The effect of divalent cations on the amidolytic activity of bovine plasma activated protein C and des-1-41-light chain activated protein C.

The effect of the divalent cations Ca2+ and Mn2+ on the amidolytic activity of bovine plasma activated protein C and a limited chymotryptic digestion product, des-1-41-light chain activated protein C, which lacks all of the gamma-carboxyglutamic acid present in activated protein C, has been examined at 30 degrees C. In each case, the enzymic activities were dependent upon the presence of these cations, which exerted their effects primarily through influence on the kcat of the reaction. For both enzymes (E), the mechanism of the reaction was most consistent with a rapid equilibrium, random addition of substrate (S), and a single cation (A), with substrate hydrolysis occurring only with the ternary complex of S.E.A. The divalent cation site of importance was not associated with gamma-carboxyglutamic acid residues and was found to be independent of the monovalent cation sites, which also function to activate the amidolytic and esterolytic activities of these enzymes.

Topographical relationships between the monovalent and divalent cation binding sites of des-1-41-light chain bovine plasma protein C and des-1-41-light chain-activated bovine plasma protein C.

The paramagnetic effect of Mn2+ on the longitudinal relaxation rate (T1)-1 of 205Tl+, when both cations are bound to des-1-41-light chain bovine plasma protein C (GDPC) and its activation product, des-1-41-light chain-activated bovine plasma protein C (GDAPC), has been assessed by 205Tl+ NMR spectroscopy. A substantial shortening of the T1 for Tl+ bound to either protein was observed in the presence of Mn2+, an effect not noted upon substitution of Mn2+ with the diamagnetic cation Ca2+, which is known to bind to these proteins in a similar fashion to Mn2+. This paramagnetic effect was employed to estimate distances between the monovalent and divalent cation sites in these proteins, approximately 6.7 +/- 0.2 A with GDPC and 8.3 +/- 0.2 A in GDAPC. These data suggest that a conformational alteration occurs upon activation of GDPC which leads to an increase in the distance between the monovalent and divalent cation sites.

205Tl+ as a spectroscopic probe of the monovalent cation binding sites of bovine plasma activated protein C and des-1-41-light-chain-activated protein C.

The kinetic properties of the stimulation by Tl+ of the amidase activity of bovine plasma activated protein C (APC) and a limited-proteolytic derivative of this enzyme, des-1-41-light chain APC (GDAPC), which has no remaining gamma-carboxyglutamic acid residues, have been compared, along with a 205Tl+ NMR analysis of the interaction of this cation with these enzymes, at 6 degrees C. In contrast to other monovalent cations, the productive kinetic complex of Tl+ and APC involves only a single Tl+ site, or class of sites, and is similar to GDAPC in this regard. In the case of each enzyme, the kinetic mechanism that best describes the participation of Tl+ is a rapid equilibrium type with random addition of the cation and substrate to the enzyme. The dissociation constants of the Tl+ X APC and Tl+ X GDAPC complexes have been determined by NMR analysis and have been found to be very similar to the same constants as calculated by kinetic means. These cation sites are also present intact on each zymogen, demonstrating that they are not generated as a result of activation. Our results also show that the Ca2+ binding sites of these proteins are exclusive of the T1+ site and that some interference with Tl+ binding is exercised by an active site-directed affinity label. We conclude that Tl+ can be effectively employed as a spectroscopic probe of the monovalent cation sites that serve an extensive stimulatory role in the amidolytic and esterolytic activities of APC.

The binding of Mn 2+ to bovine plasma protein C, des(1–41)-light chain protein C, and activated des(1–41)-light chain activated protein C

The binding isotherms of Mn 2+ to bovine plasma protein C (PC), des(1–41)-light chain protein C (GDPC), and activated GDPC (GDAPC) have been measured. PC contains 14–16 total Mn 2+ binding sites, a value that is reduced to approximately 7–8 in the presence of NaCl. The average K d of the latter sites is 230 ± 30 μ m. Upon removal of a 41-residue peptide from the amino terminus of the light chain of PC, and, concomitantly, all of the γ-carboxyglutamic acid residues, the resulting protein, GDPC, possesses a single Mn 2+ site of K d = 120 ± 20 μM. Activation of GDPC to GDAPC results in a slight lowering of the K d for the single Mn 2+ binding site to 53 ± 8 μ m, a value that is essentially unchanged in the presence of monovalent cations, a competitive inhibitor of the enzyme, or an active site directed affinity label. The Mn 2+ on GDAPC is displaced by Ca 2+, suggesting that the protein binding site for these two divalent cations is the same. These studies establish that Mn 2+ is a suitable spectroscopic probe for the Ca 2+ binding site of GDAPC, and that the divalent cation site is separate from the monovalent cation site(s) and the active site of the enzyme.

Divalent cations and the phosphatase activity of the (Na + K)-dependent ATPase.

Phosphatase activity of a kidney (Na + K)-ATPase preparation was optimally active with Mg2+ plus K+. Mn2+ was less effective and Ca2+ could not substitute for Mg2+. However, adding Ca2+ with Mg2+ or substituting Mn2+ for Mg2+ activated it appreciably in the absence of added K+, and all three divalent cations decreased apparent affinity for K+. Inhibition by Na+ decreased with higher Mg2+ concentrations, when Ca2+ was added, and when Mn2+ was substituted for Mg2+. Dimethyl sulfoxide, which favors E2 conformations of the enzyme, increased apparent affinity for K+, whereas oligomycin, which favors E1 conformations, decreased it. These observations are interpretable in terms of activation through two cases of cation sites. (i) At divalent cation sites, Mg2+ and Mn2+, favoring (under these conditions) E2 conformations, are effective, whereas Ca2+, favoring E1, is not, and monovalent cations complete. (ii) At monovalent cation sites divalent cations compete with K+, while Na+ at these sites favors E1 conformations. K+ increases the Km for substrate, but both Ca2+ and Mn2+ decrease it, perhaps by competing with K+. On the other hand, phosphatase activity in the presence of Na+ plus K+ is stimulated by dimethyl sulfoxide, by higher concentrations of Mg2+ and Mn2+, but not by adding Ca2+; this is consistent with stimulation occurring through facilitation of an E1 to E2 transition, perhaps an E1-P to E2-P step like that in the (Na + K)-ATPase reaction sequence. However, oligomycin stimulates phosphatase activity with Mg2+ plus Na+ alone or Mg2+ plus low K+: this effect of oligomycin may reflect acceleration, in the absence of adequate K+, of an alternative E2-P to E1 pathway bypassing the monovalent cation-activated steps in the hydrolytic sequence.

Interaction of valinomycin and monovalent cations with the (Ca2+,Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum.

The interactions of monovalent cations and of the K+-specific ionophore, valinomycin, with the Ca2+-ATPase of skeletal muscle of sarcoplasmic reticulum have been studied in the absence of cation gradients by their effects on enzyme turnover and on the ATP plus Ca2+-dependent enhanced fluorescence of the ATP analogue, 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)-adenosine 5'-triphosphate (TNP-ATP) (Watanabe, T., and Inesi, G. (1982) J. Biol. Chem. 257, 11510-11516). Monovalent cations decreased turnover-dependent TNP-ATP fluorescence in the series K+ greater than Rb+ approximately equal to Cs+ greater than Na+ greater than Li+ (K0.5 = 49, 73, 75, 94, and 246 mM, respectively), consistent with the known specificity of the monovalent cation binding site that stimulates turnover and E-P hydrolysis. Valinomycin (200 nmol/mg), in the absence of monovalent cations, decreased ATPase activity by 30% and abolished the stimulatory effects of 150 mM KCl or NaCl on turnover. The ionophore alone enhanced TNP-ATP fluorescence by 20% and altered the specificity and affinity of the site that inhibited TNP-ATP fluorescence to Cs+ greater than Rb+ greater than K+ approximately equal to Na+ greater than Li+ (K0.5 = 79, 111, 134, 136, and 270 mM, respectively), which follows the Hofmeister series for effectiveness of monovalent lyotropic cations. TNP-ATP binding was not affected by either monovalent cations or valinomycin. Inhibition of turnover-dependent TNP-ATP fluorescence appears to be a useful parameter for monitoring monovalent cation binding to the Ca2+-ATPase. It is concluded that the ionophore interacts directly with the Ca2+-ATPase, independent of its K+ conductance effects on the lipid bilayer, and modifies the affinity and specificity of the monovalent cation site, either by direct interaction or by the formation of a valinomycin-monovalent cation-enzyme complex.