The environment of the high-affinity cation binding site on actin and the separation between cation and ATP sites as revealed by proton NMR and fluorescence spectroscopy

The lanthanide ions Lu3+ (diamagnetic) and Gd3+ (paramagnetic broadening probe) were used to displace Ca2+ from the high-affinity cation binding site on G-actin. The effects of these higher-affinity ions on the proton nuclear magnetic resonance spectrum of actin were recorded. The aliphatic proton envelope in the Gd-actin sample exhibited a complex array of changes due to the proximity of Gd to several aliphatic residues. No such changes were observed in the diamagnetic Lu-actin control spectrum. By contrast, the aromatic proton envelope remained largely unaffected in both Gd-actin and Lu-actin samples. However, the adenosine moiety on the actin-bound ATP became increasingly mobilized without the triphosphate chain being released from the ATP binding site. Maximum adenosine mobilization occurred with approximately 1 mol of lanthanide ion bound per mol of actin. The absence of changes in the aromatic proton envelope suggests that the high-affinity cation binding site is in a region well removed from the adenosine moiety of bound ATP as well as any aromatic side-chains. The separation of the ATP and cation sites was further explored using the fluorescent ATP analogues FTP and epsilon-ATP. Tb3+ bound to the high-affinity cation site was found to be separated by 16 A from the FTP chromophore bound to the nucleotide binding site on actin. Since this distance is greater than can be accommodated on a model of the Tb-ATP complex, we conclude that the sites are physically separate. This conclusion was further reinforced by experiments involving the quenching of epsilon-ATP fluorescence by Mn2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Divalent cation binding to the high- and low-affinity sites on G-actin

Metal binding to skeletal muscle G-actin has been assessed by equilibrium dialysis using 45Ca2+ and by kinetic measurements of the increase in the fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine-labeled actin. Two classes of cation binding sites were found on G-actin which could be separated on the basis of their Ca2+ affinity: a single high-affinity site with a Kd considerably less than 1 microM and three identical moderate-affinity binding sites with a Kd of 18 microM. The data for the Mg2+-induced fluorescence enhancement of actin labeled with N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine support a previously suggested mechanism [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886] in which Ca2+ is replaced by Mg2+ at the moderate affinity site(s), followed by a slow actin isomerization. This isomerization occurs independently of Ca2+ release from the high-affinity site. The fluorescence data do not support a mechanism in which this isomerization is directly related to Ca2+ release from the high-affinity site. Fluorescence changes of labeled actin associated with adding metal chelators are complex and do not reflect the same change induced by Mg2+ addition. Fluorescence changes in the labeled actin have also been observed for the addition of Cd2+ or Mn2+ instead of Mg2+. It is proposed actin may undergo a host of subtle conformational changes dependent on the divalent cation bound. We have also developed a method by which progress curves of a given reaction can be analyzed by nonlinear regression fitting of kinetic simulations to experimental reaction time courses.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of Mg2+ at the high-affinity and low-affinity sites on the polymerization of actin and associated ATP hydrolysis

Actin contains a single high-affinity cation-binding site, for which Ca2+ and Mg2+ can compete, and multiple low-affinity cation-binding sites, which can bind Ca2+, Mg2+, or K+. Binding of cations to the low-affinity sites causes polymerization of monomeric actin with either Ca2+ or Mg2+ at the high-affinity site. A rapid conformational change occurs upon binding of cations to the low-affinity sites (G----G) which is apparently associated with the initiation of polymerization. A much slower conformational change (G----G', or G----G' if the low-affinity sites are also occupied) follows the replacement of Ca2+ by Mg2+ at the high-affinity site. This slow conformational change is reflected in a 13% increase in the fluorescence of G-actin labeled with the fluorophore 7-chloro-4-nitrobenzene-2-oxadiazole (NBD-labeled actin). The rate of the ATP hydrolysis that accompanies elongation is slower with Ca-G-actin than with Mg-G'-actin (i.e. with Ca2+ rather than Mg2+ at the high-affinity site) although their rates of elongation are similar. The slow ATP hydrolysis on Ca-F-actin causes a lag in the increase in fluorescence associated with the elongation of actin labeled with the fluorophore N-pyrene iodoacetamide (pyrenyl-labeled actin), even though there is no lag in the elongation rate, because pyrenyl-labeled ATP-F-actin subunits have a lower fluorescence intensity than pyrenyl-labeled ADP-F-actin subunits. The effects of the cation bound to the high-affinity binding site must, therefore, be considered in quantitatively analyzing the kinetics of polymerization of NBD-labeled actin and pyrenyl-labeled actin. Although their elongation rates are not very different, the rate of nucleation is much slower for Ca-G-actin than for Mg-G'-actin, probably because of the slower rate of ATP hydrolysis when Ca2+ is bound to the high-affinity site.     

Mechanism for nucleotide exchange in monomeric actin

Rabbit skeletal muscle G-actin has been treated to obtain ADP, 1,N6-ethenoadenosine diphosphate (epsilon-ADP), or 1,N6-ethenoadenosine triphosphate (epsilon-ATP) at the nucleotide binding site and either Mg2+ or Ca2+ at high- and moderate-affinity metal binding sites. Apparent rates or rate constants for the displacement of the actin-bound nucleotides by epsilon-ATP or ATP have been obtained by stopped-flow measurements at pH 8 and 20 degrees C of the fluorescence difference between bound and free epsilon-ATP or epsilon-ADP. In the presence of Ca2+, displacement of ADP by epsilon-ATP or epsilon-ADP by ATP is a biphasic process, but in the presence of low (less than 10 microM) Mg2+ concentrations, it is a slow first-order process. At high levels of Mg2+ (greater than 50 microM), low ADP concentrations displace epsilon-ATP from G-actin as a consequence of Mg2+ binding to moderate-affinity sites on the actin. Displacement of epsilon-ATP by ATP in the presence of either Ca2+ or Mg2+ is slow at low ATP concentrations, but the rate is increased by high ATP concentrations. Using ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, we find that nucleotide exchange is affected differently by the removal of Ca2+ from the high-affinity site compared to Ca2+ removal from moderate-affinity sites. A mechanism for the displacement reaction is proposed in which there are two forms of an actin-ADP complex and metal binding influences the ratio of these forms as well as the binding of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energy-transfer studies of the distance between the high-affinity metal binding site and the colchicine and 8-anilino-1-naphthalenesulfonic acid binding sites on calf brain tubulin

The high-affinity metal divalent cation Mg2+, associated with the exchangeable guanosine 5'-triphosphate (GTP) binding site (E site) on purified tubulin, has been replaced by the transition metal ion Co2+ on tubulin as well as on the tubulin-colchicine, tubulin-allocolchicine and tubulin-8-anilino-1-naphthalenesulfonic acid (tubulin-ANS) complexes. While pure native tubulin readily incorporated 0.8 atom of Co2+ per tubulin alpha-beta dimer, incorporation was reduced to 0.4 atom of Co2+ per mole of tubulin when it was complexed with colchicine, indicating that the conformational change induced in tubulin by the binding of colchicine leads to a reduced accessibility of the divalent cation binding site linked to the E site without necessarily changing the intrinsic binding constant. The fluorescence emission spectra of tubulin-bound colchicine, allocolchicine, and ANS displayed a strong overlap with the Co2+ absorption spectrum, identifying these as adequate donor-acceptor pairs. Fluorescence energy-transfer measurements were carried out between tubulin-bound colchicine (or allocolchicine) and ANS as donors and tubulin-complexed Co2+ as acceptor. It was found that the distance between the ANS and the high-affinity divalent cation binding sites is greater than 28 A, while that between the colchicine and the divalent cation binding sites is greater than 24 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Divalent cation-, nucleotide-, and polymerization-dependent changes in the conformation of subdomain 2 of actin.

Conformational changes in subdomain 2 of actin were investigated using fluorescence probes dansyl cadaverine (DC) or dansyl ethylenediamine (DED) covalently attached to Gln41. Examination of changes in the fluorescence emission spectra as a function of time during Ca2+/Mg2+ and ATP/ADP exchange at the high-affinity site for divalent cation-nucleotide complex in G-actin confirmed a profound influence of the type of nucleotide but failed to detect a significant cation-dependent difference in the environment of Gln41. No significant difference between Ca- and Mg-actin was also seen in the magnitude of the fluorescence changes resulting from the polymerization of these two actin forms. Evidence is presented that earlier reported cation-dependent differences in the conformation of the loop 38-52 may be related to time-dependent changes in the conformation of subdomain 2 in DED- or DC-labeled G-actin, accelerated by substitution of Mg2+ for Ca2+ in CaATP-G-actin and, in particular, by conversion of MgATP- into MgADP-G-actin. These spontaneous changes are associated with a denaturation-driven release of the bound nucleotide that is promoted by two effects of DED or DC labeling: lowered affinity of actin for nucleotide and acceleration of ATP hydrolysis on MgATP-G-actin that converts it into a less stable MgADP form. Evidence is presented that the changes in the environment of Gln41 accompanying actin polymerization result in part from the release of Pi after the hydrolysis of ATP on the polymer. A similarity of this change to that accompanying replacement of the bound ATP with ADP in G-actin is discussed.     

Role of ATP-bound divalent metal ion in the conformation and function of actin. Comparison of Mg-ATP, Ca-ATP, and metal ion-free ATP-actin

The fluorescence of N-acetyl-N'-(sulfo-1-naphthyl)ethylenediamine (AEDANS) covalently bound to Cys-374 of actin is used as a probe for different conformational states of G-actin according to whether Ca-ATP, Mg-ATP, or unchelated ATP is bound to the nucleotide site. Upon addition of large amounts (greater than 10(2)-fold molar excess) of EDTA to G-actin, metal ion-free ATP-G-actin is obtained with EDTA bound. Metal ion free ATP-G-actin is characterized by a higher AEDANS fluorescence than Mg-ATP-G-actin, which itself has a higher fluorescence than Ca-ATP-G-actin. Evidence for EDTA binding to G-actin is shown using difference spectrophotometry. Upon binding of EDTA, the rate of dissociation of the divalent metal ion from G-actin is increased (2-fold for Ca2+, 10-fold for Mg2+) in a range of pH from 7.0 to 8.0. A model is proposed that quantitatively accounts for the kinetic data. The affinity of ATP is weakened 10(6)-fold upon removal of the metal ion. Metal ion-free ATP-G-actin is in a partially open conformation, as indicated by the greater accessibility of -SH residues, yet it retains functional properties of polymerization and ATP hydrolysis that appear almost identical to those of Ca-ATP-actin, therefore different from those of Mg-ATP-actin. These results are discussed in terms of the role of the ATP-bound metal ion in actin structure and function.     

Stability and dynamics of G-actin: back-door water diffusion and behavior of a subdomain 3/4 loop.

Molecular dynamics simulations have been performed on solvated G-actin bound to ADP and ATP, starting with the crystal structure of the actin-DNase 1 complex, including a Ca2+ or Mg2+ ion at the high-affinity divalent cation-binding site. Water molecules have been found to enter the nucleotide-binding site (phosphate vicinity) along two pathways, from the side where the nucleotide base is exposed to water, as well as from the opposite side. The water channels suggest a "back-door" mechanism for ATP hydrolysis in which the phosphate is released to a side opposite that of nucleotide binding and unbinding. The simulations also reveal a propensity of G-actin to alter its crystallographic structure toward the filamentous structure. Domain movement closes the nucleotide cleft, the movement being more pronounced for bound Mg2+. The conformational change is interpreted as a response of the system to missing water molecules in the crystal structure. The structures arising in the simulations, classified according to nucleotide cleft separation and radius of gyration of the protein, fall into two distinct clusters: a cluster of states that are similar to the G-actin crystal structure, and a cluster of states with small cleft separation and with the subdomain 3/4 loop 264-273 detached from the protein. The latter states resemble the putative filamentous structure of actin, in which the loop connects the two strands of the actin filament.     

Effects of various amino acid replacements on the conformational stability of G-actin

Circular dichroic spectra of native, EDTA-treated and heat-denatured G-actin from chicken gizzard smooth muscle are virtually the same as those of rabbit skeletal muscle actin. The rates of changes produced by EDTA or heat in the secondary structure are, however, higher in the case of gizzard actin. Similar differences were found in the rates of inactivation as measured by loss of polymerizability during incubation with EDTA or Dowex 50. The results are explicable in terms of local differences in the conformation at specific site(s) important for maintaining the native state of actin monomer. Involvement of the ATP binding site was shown by measuring the equilibrium constant for the binding of ATP to the two actins. Difference in the conformation of some additional site(s) is indicated by a higher rate constant of inactivation of nucleotide-free actin observed for gizzard actin. No significant difference was found in the equilibrium constant for the binding of Ca2+ at the single high-affinity site in gizzard and skeletal muscle actin. Comparison of inactivation kinetics of actin from chicken gizzard, rabbit skeletal, bovine aorta, and bovine cardiac muscle suggests that the amino acid replacements Val-17----Cys-17 and/or Thr-89----Ser-89 have a destabilizing effect on the native conformation of G-actin. The results indicate that deletion of the acidic residue at position 1 of the amino acid sequence has no effect on the conformation of the ATP binding site and the high-affinity site for divalent cation as well.     

Evidence for the direct interaction between tightly bound divalent metal ion and ATP on actin. Binding of the lambda isomers of beta gamma-bidentate CrATP to actin

Competition between Ca2+ and Mg2+ for binding to a single high affinity site on actin has been confirmed. Occupancy of this site only by either Ca2+ or Mg2+ affects the conformation of actin and its ability to form nuclei and hydrolyze ATP. G-actin binds the beta gamma-bidentate CrATP, a substitution inert analog of metal-ATP complexes, and shows a high specificity for the lambda isomers. Binding of CrATP to ADP-actin is accompanied by the dissociation of tightly bound ADP and Ca2+. CrATP-actin shows a high tendency to form nuclei, like MgATP-actin. Polymerization of CrATP-actin is accompanied by cleavage of the gamma-phosphate, but subsequent Pi release cannot occur because the product of the reaction is the stable CrADP-Pi complex. All these results support the view that the divalent metal ion tightly bound to actin interacts with the beta- and gamma-phosphates of ATP in the nucleotide site.     

Occlusion of divalent cations in the phosphorylated calcium pump of sarcoplasmic reticulum

The calcium pump of sarcoplasmic reticulum possesses high-affinity calcium-binding and ATP-binding sites. At 0 degrees C pH 6.8 and in the absence of calcium, about 3.5 nmol/mg of high-affinity ATP-binding sites are titrated with a dissociation constant, Kd, of 5 microM. In the presence of Ca2+, ATP phosphorylates the enzyme at a much lower concentration: K 1/2 = 100 nM. In the absence of ATP the calcium ions reversibly bind to the high-affinity calcium sites (6.5 nmol/mg); however the following is shown in this paper. 1. Phosphorylation of the enzyme in the presence of calcium leads to the immediate occlusion of the calcium ions bound to the high-affinity sites. 2. Two moles of calcium are occluded per mole of phosphoenzyme formed. 3. Occlusion can be reversed by ADP. 4. Transport is a slower process which occurs in the presence of Mg2+ at the same rate as the spontaneous decay of the phosphoenzyme. Experiments performed in the absence of magnesium reveal another divalent cation binding site which is probably directly involved in ATP and Pi binding. The nature of the cation bound to this site determines the stability and ADP-sensitivity of the phosphoenzyme.     

Crystalline actin tubes. I. Is the conformation of the lanthanide-induced actin tube monomer more like F-actin than G-actin?

Actin, isolated from rabbit skeletal muscle, forms highly-ordered aggregates when it binds six moles of the lanthanide ion, Gd3+. In the presence of 0.1 M KCl, these aggregates are referred to as actin tubes. The monomer contained in the repeating subunit of these tubes possess a number of functional characteristics which include: (i) binding to myosin or subfragment-1 of myosin; (ii) rapid conversion into filamentous Gd-actin which can activate myosin ATPase activity; (iii) a slow rate of exchange of the bound nucleotide; (iv) a slow rate of exchange of the metal cation; (v) a resistance to digestion by proteolytic enzymes. Additionally, the monomer of the Gd-actin tube structures appears to stoichiometrically bind ATP and exhibit a lower minimum protein concentration for tube formation than is needed for the formation of F-actin. The properties listed above suggest that the actin monomer, which comprises the Gd-actin tubes, bears little resemblance to either the G-actin monomer or the recently-described actin monomer conformation that exists under conditions that favour polymerization. The data suggest that the actin molecules which comprise the Gd-actin tube structures contain sites which bind myosin, nucleotide and metal cations and that these sites are similar to the sites on F-actin.     

Changes in conformation and nucleotide binding of Ca, Mn, or MgG-actin upon removal of the bound divalent cation. Studies of ultraviolet difference spectra and optical rotation

The ultraviolet difference spectra of EDTA-induced denaturation of dithiothreitoltreated actin prepared with either Ca²⁺, Mn²⁺, or Mg²⁺ as the strongly bound cation showed no appreciable difference, nor could any difference be found in the change of optical rotation. However, at different wavelengths the changes in the spectra have different rates and these rates do differ significantly depending on the bivalent cation bound to G-actin. The nucleotide and the cation appear to be removed simultaneously and at the fastest rate; about 50–80% is released within 1 min. The spectral changes have two phases: a fast change whose detailed kinetics have not been investigated in this paper, followed by a slower rate with first-order kinetics. The changes of optical rotation follow a single-phase first-order kinetics. The rates depend on the divalent cation, the sequence being Mn²⁺ > Ca²⁺ > Mg²⁺. ATP release is partially reversible upon Ca²⁺ addition; the reversibility is diminished as the time of incubation with EDTA is increased. On rebinding of ATP and Ca²⁺, the spectral and optical rotatory changes are not reversed, but no further changes occur. Such an EDTA-treated actin is polymerizable after addition of Ca²⁺, and the G-actin obtained after polymerization and depolymerization shows the same spectral change on a second addition of EDTA as the original actin. On the basis of these observations a scheme is suggested for the denaturation of G-actin.     

Nd3+ and Co2+ binding to sarcoplasmic reticulum CaATPase. An estimation of the distance from the ATP binding site to the high-affinity calcium binding sites

Nd3+ binding to sarcoplasmic reticulum (SR) was detected by inhibition of ATPase activity and directly by a fluorimetric assay. Both methods indicated that Nd3+ inhibited the ATPase activity by binding in the high-affinity Ca2+ binding sites. The stoichiometry of binding was about 11 nmol of Nd3+ bound per mg of SR proteins at pNd = 6.5. At higher [Nd3+], substantial nonspecific binding occurred. The association constant for Nd3+ binding to the high-affinity Ca2+ binding sites was estimated to be near 2 X 10(9) M-1. When the CaATPase was inactivated with fluorescein isothiocyanate (FITC), 5.3 nmol were bound per mg of SR protein. This fluorescent probe is known to bind in the ATP binding site. The stoichiometry of Nd3+ binding to FITC-labeled CaATPase was the same, within experimental error, as to the unlabeled CaATPase. Fluorescence energy transfer between FITC in the ATP site and Nd3+ in the Ca2+ sites was found to be very small. This donor-acceptor pair has a critical distance of 0.93 nm and the distance between the ATP site and the closest Ca2+ was estimated to be greater than 2.1 nm. Parallel measurements with FITC-labeled SR and Co2+, an acceptor with a critical distance 1.2 nm, suggested the ATP and Ca2+ binding sites are greater than 2.6 nm apart.     

Dual divalent cation requirement for activation of pyruvate kinase; essential roles of both enzyme- and nucleotide-bound metal ions.

Rabbit muscle pyruvate kinase requires two divalent cations per active site for catalysis of the enolization of pyruvate in the presence of adenosine 5'-triphosphate (ATP). One divalent cation is bound directly to the enzyme and forms a second sphere complex with the bound ATP (site 1). The second divalent cation is directly coordinated to the phosphoryl groups of ATP and does not interact with the enzyme (site 2). The essential role of the divalent cation at site 1 is shown by the requirement for Mg2+ or Mn2+ for the enolization of pyruvate in the presence of the substitution inert Cr3+-ATP complex. The rate of detritiation of pyruvate shows a hyperbolic dependence of Mn2+ concentration in the presence of high concentrations of enzyme and Cr3+-ATP. A dissociation constant for Mn2+ from the pyruvate kinase-Mn2+-ATP-Cr3+-pyruvate complex of 1.3 +/- 0.5 muM is determined by the kinetics of detritiation of pyruvate and by parallel Mn2+ binding studies using electron paramagnetic resonance. The essential role of the divalent cation at site 2 is shown by the sigmoidal dependence of the rate of detritiation of pyruvate on Mn2+ concentration in the presence of high concentrations of enzyme and ATP yielding a dissociation constant of 29 +/- 9 muM for Mn2+ from site 2. This value is similar to the dissociation constant of the binary Mn-ATP complex (14 +/- 6 muM) determined under similar conditions. The rate of detritiation of pyruvate is proportional to the concentration of the pyruvate kinase-Mn2+-ATP-Mn2+-pyruvate complex, as determined by parellel kinetic and binding studies. Variation of the nature of the divalent cation at site 1 in the presence of CrATP causes only a twofold change in the rate of detritiation of pyruvate which does not correlate with the pKa of the metal-bound water. Variation of the nature of the divalent cation at both sites in the presence of ATP causes a sevenfold variation in the rate of detritiation or pyruvate that correlates with the pKa of the metal-bound water. The greater rate of enolization observed with CrATP fits this correlation, indicating that the electrophilicity of the nucleotide bound metal (at site 2) determines the rate of enolization of pyruvate.     

Metal requirements of a diadenosine pyrophosphatase from Bartonella bacilliformis: magnetic resonance and kinetic studies of the role of Mn2+

Recombinant IalA protein from Bartonella bacilliformis is a monomeric adenosine 5'-tetraphospho-5'-adenosine (Ap4A) pyrophosphatase of 170 amino acids that catalyzes the hydrolysis of Ap4A, Ap5A, and Ap6A by attack at the delta-phosphorus, with the departure of ATP as the leaving group [Cartwright et al. (1999) Biochem. Biophys. Res. Commun. 256, 474-479]. When various divalent cations were tested over a 300-fold concentration range, Mg2+, Mn2+, and Zn2+ ions were found to activate the enzyme, while Ca2+ did not. Sigmoidal activation curves were observed with Mn2+ and Mg2+ with Hill coefficients of 3.0 and 1.6 and K0.5 values of 0.9 and 5.3 mM, respectively. The substrate M2+ x Ap4A showed hyperbolic kinetics with Km values of 0.34 mM for both Mn2+ x Ap4A and Mg2+ x Ap4A. Direct Mn2+ binding studies by electron paramagnetic resonance (EPR) and by the enhancement of the longitudinal relaxation rate of water protons revealed two Mn2+ binding sites per molecule of Ap4A pyrophosphatase with dissociation constants of 1.1 mM, comparable to the kinetically determined K0.5 value of Mn2+. The enhancement factor of the longitudinal relaxation rate of water protons due to bound Mn2+ (epsilon b) decreased with increasing site occupancy from a value of 12.9 with one site occupied to 3.3 when both are occupied, indicating site-site interaction between the two enzyme-bound Mn2+ ions. Assuming the decrease in epsilon(b) to result from cross-relaxation between the two bound Mn2+ ions yields an estimated distance of 5.9 +/- 0.4 A between them. The substrate Ap4A binds one Mn2+ (Kd = 0.43 mM) with an epsilon b value of 2.6, consistent with the molecular weight of the Mn2+ x Ap4A complex. Mg2+ binding studies, in competition with Mn2+, reveal two Mg2+ binding sites on the enzyme with Kd values of 8.6 mM and one Mg2+ binding site on Ap4A with a Kd of 3.9 mM, values that are comparable to the K0.5 for Mg2+. Hence, with both Mn2+ and Mg2+, a total of three metal binding sites were found-two on the enzyme and one on the substrate-with dissociation constants comparable to the kinetically determined K0.5 values, suggesting a role in catalysis for three bound divalent cations. Ca2+ does not activate Ap4A pyrophosphatase but inhibits the Mn2+-activated enzyme competitively with a Ki = 1.9 +/- 1.3 mM. Ca2+ binding studies, in competition with Mn2+, revealed two sites on the enzyme with dissociation constants (4.3 +/- 1.3 mM) and one on Ap4A with a dissociation constant of 2.1 mM. These values are similar to its Ki suggesting that inhibition by Ca2+ results from the complete displacement of Mn2+ from the active site. Unlike the homologous MutT pyrophosphohydrolase, which requires only one enzyme-bound divalent cation in an E x M2+ x NTP x M2+ complex for catalytic activity, Ap4A pyrophosphatase requires two enzyme-bound divalent cations that function in an active E x (M2+)2 x Ap4A x M2+ complex.     

An electron paramagnetic resonance study of bovine alpha-lactalbumin-metal ion complexes

alpha-lactalbumin has at least three distinct cation binding regions: a Ca(II)-Gd(III) site, a Cu(II)-Zn(II) site and a VO2+ site as observed from electron paramagnetic resonance (EPR) studies of complexes with the bovine protein. Gadolinium, which bound to the calcium site of the protein with a subnanomolar dissociation constant, yielded EPR spectra at 9.5 GHz (X-band) that exhibited features from g = 8 to g = 2. At 35 GHz (Q-band) the central fine structure transition (Ms = 1/2----Ms = -1/2) gave a well-defined powder pattern. The zero-field splitting was large, as reflected in the second-order splitting of the central fine structure transition of about 1 kG. There was also evidence for additional, low affinity binding site(s) for Gd(III). Addition of either Zn(II) or Al(III) did not affect the amplitudes or positions of the bound Gd(III) EPR spectrum. The Cu(II)-alpha-lactalbumin complex gave a typical axially symmetric spectrum (g parallel = 2.260, g perpendicular = 2.056, A parallel = 171 G) with a partially resolved superhyperfine interaction attributable to at least one directly coordinated nitrogen ligand. Addition of Cu(II) to Gd(III)-alpha-lactalbumin gave an EPR spectrum that was a superposition of signals from the individual Gd(III)- and Cu(II)-alpha-LA spectra. The absence of any magnetic interactions in the Gd(III)-Cu(II)-alpha-lactalbumin species indicated that the two cation sites were more than 10 A apart. On the other hand, addition of Zn(II) to Cu(II)-alpha-lactalbumin gave a set of EPR lines due to free or loosely bound Cu(II), confirming that the Cu(II) was displaced by zinc.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lithium-7 nuclear magnetic resonance, water proton nuclear magnetic resonance, and gadolinium electron paramagnetic resonance studies of the sarcoplasmic reticulum calcium ion transport adenosine triphosphatase.

The interactions of gadolinium ion, lithium, and two substrate analogues, beta,gamma-imido-ATP (AMP-PNP) and tridentate CrATP, with the calcium ion transport adenosine triphosphatase (Ca2+-ATPase) of rabbit muscle sarcoplasmic reticulum have been examined by using 7Li+ NMR, water proton NMR, and Gd3+ EPR studies. Steady-state phosphorylation studies indicate that Gd3+ binds to the Ca2+ activator sites on the enzyme with an affinity which is approximately 10 times greater than that of Ca2+. 7Li+, which activates the Ca2+-ATPase in place of K+, has been found to be a suitable nucleus for probing the active sites of monovalent cation-requiring enzymes. 7Li+ nuclear relaxation studies demonstrate that the binding of Gd3+ ion to the two Ca2+ sites on Ca2+-ATPase increases the longitudinal relaxation rate (1/T1) of enzyme-bound Li+. The increase in 1/T1 was not observed in the absence of enzyme, indicating that the ATPase enhances the parmagnetic effect of Gd3+ on 1/T1 of 7Li+. Water proton relaxation studies also show that the ATPase binds Gd3+ at two tight-binding sites. Titrations of Gd3+ solutions with Ca2+-ATPase indicate that the tighter of the two Gd3+-binding sites (site 1) provides a ghigher enhancement of water relaxation than the other, weaker Gd3+ site (site 2) and also indicate that the average of the enhancements at the two sites is 7.4. These data, together with a titration of the ATPase with Gd3+ ion, yield enhancements, epsilonB, of 9.4 at site 1 and 5.4 at site 2. Analysis of the frequency dependence of 1/T1 of water indicates that the electron spin relaxation taus of Gd3+ is unusually long (2 X 10(-9) s) and suggests that the Ca2+-binding sites on the ATPase experience a reduced accessiblity of solvent water. This may indicate that the Ca2+ sites on the Ca2+-ATPase are buried or occluded within a cleft or channel in the enzyme. The analysis of the frequency dependence is also consistent with three exchangeable water protons on Gd3+ at site 1 and two fast exchanging water protons at site 2. Addition of the nonhydrolyzing substrate analogues, AMP-PNP and tridenate CrATP, to the enzyme-Gd3+ complex results in a decrease in the observed enhancement, with little change in the dipolar correlation time for Gd3+, consistent with a substrate-induced decrease in the number of fast-exchanging water protons on enzyme-bound Gd3+. From the effect of Gd3+ on 1/T1 of enzyme-bound Li+, Gd3+-Li+ separations of 7.0 and 9.1 A are calculated. On the assumption of a single Li+ site on the enzyme, these distances set an upper limit on the separation between Ca2+ sites on the enzyme of 16.1 A.     

pH-induced changes in G-actin conformation and metal affinity

Metal-induced conformational changes in actin at 20 degrees C have been investigated as a function of pH using actin labeled at Cys-374 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine. At pH 8, the addition of a high Ca2+ concentration (2 mM) to G-actin gives an instantaneous fluorescence increase while the addition of a high Mg2+ concentration gives both an instantaneous and a slow fluorescence increase. The instantaneous increase is interpreted as divalent cation binding to low-affinity, relatively nonspecific sites, while the slow response is attributed to Mg2+ binding to specific sites of moderate affinity [Zimmerle, C.T., Patane, K., & Frieden, C. (1987) Biochemistry 26, 6545-6552]. The magnitudes of both the instantaneous and slow fluorescence increases associated with Mg2+ addition to G-actin are shown here to decrease as the pH is lowered while the fluorescence of labeled G-actin in the presence of low or moderate Ca2+ concentrations (less than 200 microM) increases. The pH-dependent data suggest that protonation of a single class of residues with an approximate pK of 6.8 alters the immediate environment of the label differently depending upon the cation bound at the moderate-affinity site. The pH-dependent changes in the magnitude of the slow fluorescence response upon Mg2+ addition to Ca2+-actin are not associated with changes in the Mg2+ affinity at the moderate-affinity site but result from protonation altering the fluorescence response to Mg2+ binding. Protonation of this same class of residues is proposed to induce an actin conformation similar to that induced by cation binding at the low-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of di- and trivalent metal ions on the binding of [3H-Tyr1, D-Ala2, D-Leu5] enkephalin to opiate receptors from the rat brain

The influence of Ca2+, Mg2+, Mn2+, Sr2+, La3+, Nd3+, Sm3+, Eu3+, and Gd3+ ions on the binding of labeled, stable enkephalin analogue, [3H-Tyr1, D-Ala2, D-Leu5]enkephalin, to opiate receptors of the rat brain membrane preparations has been investigated. The formation of the complex can be described by a scheme involving at least two independent binding sites. The high affinity site does not discriminate the divalent and trivalent metal ions: all examined cations enhanced the enkephalin affinity for this site. The ligand binding to the low affinity site is potentiated only by Mn2+, Mg2+, and lathanoides. The maximal concentration of the binding sites of the above two types is not affected by the cations. The increase in the ionic strength of the solution entails a decrease in the affinity of the ligand for the high affinity binding site. It is shown that the effect of both di- and trivalent metal cations on the [3H-Tyr1, D-Ala2, D-Leu3] enkephalin binding is mediated through one cation attachment site on the respective enkephalin receptor.