Evidence for two conformational states of thioredoxin reductase from Escherichia coli: use of intrinsic and extrinsic quenchers of flavin fluorescence as probes to observe domain rotation.

Thioredoxin reductase (TrxR) from Escherichia coli consists of two globular domains connected by a two-stranded beta sheet: an FAD domain and a pyridine nucleotide binding domain. The latter domain contains the redox-active disulfide composed of Cys 135 and Cys 138. TrxR is proposed to undergo a conformational change whereby the two domains rotate 66 degrees relative to each other (Waksman G, Krishna TSR, Williams CH Jr, Kuriyan J, 1994, J Mol Biol 236:800-816), placing either redox active disulfide (FO conformation) or the NADPH binding site (FR conformation) adjacent to the flavin. This domain rotation model was investigated by using a Cys 138 Ser active-site mutant. The flavin fluorescence of this mutant is only 7% that of wild-type TrxR, presumably due to the proximity of Ser 138 to the flavin in the FO conformation. Reaction of the remaining active-site thiol, Cys 135, with phenylmercuric acetate (PMA) causes a 9.5-fold increase in fluorescence. Titration of the PMA-treated mutant with the nonreducing NADP(H) analogue, 3-aminopyridine adenine dinucleotide phosphate (AADP+), results in significant quenching of the flavin fluorescence, which demonstrates binding adjacent to the FAD, as predicted for the FR conformation. Wild-type TrxR, with or without PMA treatment, shows similar quenching by AADP+, indicating that it exists mostly in the FR conformer. These findings, along with increased EndoGluC protease susceptibility of PMA-treated enzymes, agree with the model that the FO and FR conformations are in equilibrium. PMA treatment, because of steric limitations of the phenylmercuric adduct in the FO form, forces the equilibrium to the FR conformer, where AADP+ binding can cause fluorescence quenching and conformational restriction favors proteolytic susceptibility.     

Mechanism of coenzyme binding to human methionine synthase reductase revealed through the crystal structure of the FNR-like module and isothermal titration calorimetry

Human methionine synthase reductase (MSR) is a 78 kDa flavoprotein that regenerates the active form of cobalamin-dependent methionine synthase (MS). MSR contains one FAD and one FMN cofactor per polypeptide and functions in the sequential transfer of reducing equivalents from NADPH to MS via its flavin centers. We report the 1.9 A crystal structure of the NADP+-bound FNR-like module of MSR that spans the NADP(H)-binding domain, the FAD-binding domain, the connecting domain, and part of the extended hinge region, a feature unique to MSR. The overall fold of the protein is similar to that of the corresponding domains of the related diflavin reductase enzymes cytochrome P450 reductase and neuronal nitric oxide synthase (NOS). However, the extended hinge region of MSR, which is positioned between the NADP(H)/FAD- and FMN-binding domains, is in an unexpected orientation with potential implications for the mechanism of electron transfer. Compared with related flavoproteins, there is structural variation in the NADP(H)-binding site, in particular regarding those residues that interact with the 2'-phosphate and the pyrophosphate moiety of the coenzyme. The lack of a conserved binding determinant for the 2'-phosphate does not weaken the coenzyme specificity for NADP(H) over NAD(H), which is within the range expected for the diflavin oxidoreductase family of enzymes. Isothermal titration calorimetry reveals a binding constant of 37 and 2 microM for binding of NADP+ and 2',5'-ADP, respectively, for the ligand-protein complex formed with full-length MSR or the isolated FNR module. These values are consistent with Ki values (36 microM for NADP+ and 1.4 microM for 2',5'-ADP) obtained from steady-state inhibition studies. The relatively weaker binding of NADP+ to MSR compared with other members of the diflavin oxidoreductase family might arise from unique electrostatic repulsive forces near the 5'-pyrophosphate moiety and/or increased hydrophobic stacking between Trp697 and the re face of the FAD isoalloxazine ring. Small structural permutations within the NADP(H)-binding cleft have profound affects on coenzyme binding, which likely retards catalytic turnover of the enzyme in the cell. The biological implications of an attenuated mechanism of MS reactivation by MSR on methionine and folate metabolism are discussed.     

Kinetic mechanism of cytochrome P450 reductase from the house fly (Musca domestica).

Recombinant house fly (Musca domestica) cytochrome P450 reductase has been purified by anion exchange and affinity chromatography. Steady-state kinetics of cytochrome c reductase activity revealed a random Bi-Bi mechanism with formation of a ternary P450 reductase-NADPH-electron acceptor complex as catalytic intermediate. NADP(H) binding is essential for fast hydride ion transfer to FAD, as well as for electron transfer from FMN to cytochrome c. Reduced cytochrome c had no effect on the enzyme activity, while NADP+ and 2'-AMP inhibited P450 reductase competitively with respect to NADPH and noncompetitively with respect to cytochrome c. The affinity of the P450 reductase to NADPH is 10 times higher than to NADP+ (Kd of 0.31 and 3.3 microM, respectively). Such an affinity change during catalysis could account for a +30 mV shift of the redox potential of FAD. Cys560 was substituted for Tyr by site-directed mutagenesis. This mutation decreased enzyme affinity to NADPH 35-fold by decreasing the bimolecular rate constant of nucleotide binding with no detectable effect on the kinetic mechanism. The affinity of the C560Y mutant enzyme to NADP+ decreased 9-fold compared to the wild-type enzyme, while the affinity to 2'-AMP was not significantly affected, suggesting that Cys560 is located in the nicotinamide binding site of the active, full-size enzyme in solution.     

Characterization of the cation-binding properties of porcine neurofilaments

In the presence of physiological levels of Na+ (10 mM), K+ (150 mM), and Mg2+ (2 mM), dephosphorylated neurofilaments contained two Ca2+ specific binding sites with Kd = 11 microM per unit consisting of eight low, three middle, and three high molecular subunits, as well as 46 sites with Kd = 620 microM. Only one class of 126 sites with Kd = 740 microM was detected per unit of untreated neurofilaments. A chymotryptic fraction enriched in the alpha-helical domains of neurofilament subunits contained one high-affinity Ca2+-binding site (Kd = 3.6 microM) per domain fragment of approximately 32 kDa. This site may correspond to a region in coil 2b of the alpha-helical domain, which resembles the I-II Ca2+-binding site in intestinal Ca2+-binding protein. Homopolymeric filaments composed of the low or middle molecular weight subunits contained low-affinity Ca2+-binding sites with Kd = 37 microM and 24 microM, respectively, while the Kd values for the low-affinity sites in heteropolymeric filaments were 8-10-fold higher. Competitive binding studies, using the chymotryptic fraction to assay the high-affinity Ca2+-binding sites and 22Na+ to monitor binding to the phosphate-containing low-affinity sites, yielded Kd values for Al3+ of 0.01 microM and 4 microM, respectively. This suggests that the accumulation of Al3+ in neurons may be due in part to its binding to neurofilaments.     

Reactions of the Neurospora crassa nitrate reductase with NAD(P) analogs.

The assimilatory NADPH-nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.3) from Neurospora crassa is competitively inhibited by 3-aminopyridine adenine dinucleotide (AAD) and 3-aminopyridine adenine dinucleotide phosphate (AADP) which are structural analogs of NAD and NADP, respectively. The amino group of the pyridine ring of AAD(P) can react with nitrous acid to yield the diazonium derivative which may covalently bind at the NAD(P) site. As a result of covalent attachment, diazotized AAD(P) causes time-dependent irreversible inactivation of nitrate reductase. However, only the NADPH-dependent activities of the nitrate reductase, i.e. the overall NADPH-nitrate reductase and the NADPH-cytochrome c reductase activities, are inactivated. The reduced methyl viologen- and reduced FAD-nitrate reductase activities which do not utilize NADPH are not inhibited. This inactivation by diazotized AADP is prevented by 1 mM NADP. The inclusion of 1 muM FAD can also prevent inactivation, but the FAD effect differs from the NADP protection in that even after removal of the exogenous FAD by extensive dialysis or Sephadex G-25 filtration chromatography, the enzyme is still protected against inactivation. The FAD-generated protected form of nitrate reductase could again be inactivated if the enzyme was treated with NADPH, dialyzed to remove the NADPH, and then exposed to diazotized AADP. When NADP was substituted for NADPH in this experiment, the enzyme remained in the FAD-protected state. Difference spectra of the inactivated nitrate reductase demonstrated the presence of bound AADP, and titration of the sulfhydryl groups of the inactivated enzyme revealed that a loss of accessible sulfhydryls had occurred. The hypothesis generated by these experiments is that diazotized AADP binds at the NADPH site on nitrate reductase and reacts with a functional sulfhydryl at the site. FAD protects the enzyme against inactivation by modifying the sulfhydryl. Since NADPH reverses this protection, it appears the modifications occurring are oxidation-reduction reactions. On the basis of these results, the physiological electron flow in the nitrate reductase is postulated to be from NADPH via sulfhydryls to FAD and then the remainder of the electron carriers as follows: NADPH leads to -SH leads to FAD leads to cytochrome b-557 leads to Mo leads to NO-3.     

Eosin, a fluorescent marker for the high-affinity ATP site of (K+ + H+)-ATPase

Eosin has been used as a fluorescent probe for studying conformational states in (K+ + H+)-ATPase. The eosin fluorescence level is increased by Mg2+ (K0.5 = 0.2 mM). This increase is counteracted by K+ (I0.5 = 1.3 mM) and choline (I0.5 = 17.2 mM) and by ATP. Binding studies with eosin indicate that the increase and decrease in fluorescence is due to changes in binding of eosin to the enzyme. The Mg2+-induced specific binding has a Kd of 0.7 microM and a maximal capacity of 3.5 nmol per mg enzyme, which is equivalent to 2.5 site per phosphorylation site. These experiments and the fact that eosin competitively inhibits (K+ + H+)-ATPase towards ATP, suggest that eosin binds to ATP binding sites.     

The three conformations of the anticodon loop of yeast tRNA(Phe)

The complex conformational states of the anticodon loop of yeast tRNA(Phe) which we had previously studied with relaxation experiments by monitoring fluorescence of the naturally occurring Wye base, are analyzed using time and polarization resolved fluorescence measurements at varying counterion concentrations. Synchrotron radiation served as excitation for these experiments, which were analyzed using modulating functions and global methods. Three conformations of the anticodon loop are detected, all three occurring in a wide range of counterion concentrations with and without Mg2+, each being identified by its typical lifetime. The fluorescence changes brought about by varying the ion concentrations, previously monitored by steady state fluorimetry and relaxation methods, are changes in the population of these three conformational states, in the sense of an allosteric model, where the effectors are the three ions Mg2+, Na+ and H+. The population of the highly fluorescent M conformer (8ns), most affine to magnesium, is thus enhanced by that ligand, while the total fluorescence decreases as lower pH favors the H+-affine H conformer (0.6ns). Na+-binding of the N conformer (4ns) is responsible for complex fluorescence changes. By iterative simulation of this allosteric model the equilibrium and binding constants are determined. In turn, using these constants to simulate equilibrium fluorescence titrations reproduces the published results.     

Ferredoxin:NADP+ oxidoreductase. Equilibria in binary and ternary complexes with NADP+ and ferredoxin

Ferredoxin:NADP+ oxidoreductase (ferredoxin: NADP+ reductase, EC 1.18.1.2) was shown to form a ternary complex with its substrates ferredoxin (Fd) and NADP(H), but the ternary complex was less stable than the separate binary complexes. Kd for oxidized binary Fd-ferredoxin NADP+ reductase complex was less than 50 nM; Kd(Fd) increased with NADP+ concentration, approaching 0.5-0.6 microM when the flavoprotein was saturated with NADP+ K(NADP+) also increased from about 14 microM to about 310 microM, on addition of excess Fd. The changes in Kd were consistent with negative cooperativity between the associations of Fd and NADP+ and with our unpublished observations which suggest that product dissociation is rate-limiting in the reaction mechanism. Similar interference in binding was observed in more reduced states; NADPH released much ferredoxin:NADP+ reductase from Fd-Sepharose whether the proteins were initially oxidized or reduced. Complexation between Fd and ferredoxin: NADP+ reductase was found to shield each center from paramagnetic probes; charge specificity suggested that the active sites of Fd and ferredoxin:NADP+ reductase were, respectively, negatively and positively charged.     

Purification and properties of pyruvate dehydrogenase phosphatase from bovine heart and kidney

Pyruvate dehydrogenase phosphatase was purified to apparent homogeneity from bovine heart and kidney mitochondria. The phosphatase has a sedimentation coefficient (S20,w) of about 7.4 S and a molecular weight (Mr) of about 150 000 as determined by sedimentation equilibrium and by gel-permeation chromatography. The phosphatase consists of two subunits with molecular weights of about 97 000 and 50 000 as estimated by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Phosphatase activity resides in the Mr 50 000 subunit, which is sensitive to proteolysis. The phosphatase contains approximately 1 mol of flavin adenine dinucleotide (FAD) per mol of protein of Mr 150 000. FAD is apparently associated with the Mr 97 000 subunit. The function of this subunit remains to be established. The phosphatase binds 1 mol of Ca2+ per mol of enzyme of Mr 150 000 at pH 7.0, with a dissociation constant (Kd) of about 35 microM as determined by flow dialysis. Use of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate (EGTA) at pH 7.6 in conjunction with flow dialysis gave a Kd value for Ca2+ of about 8 microM. In the presence of both the phosphatase and the dihydrolipoyl transacetylase (E2) core of the pyruvate dehydrogenase complex, two equivalent and apparently non-interacting CA2+-binding sites were detected per unit of Mr 150 000, with a Kd value of about 24 microM in the absence and about 5 microM in the presence of EGTA. In the presence of 0.2 M KCl, which inhibits phosphatase activity about 95%, the phosphatase exhibited only one Ca2+-binding site, even in the presence of E2. The phosphatase apparently possesses an "intrinsic" Ca2+-binding site, and a second Ca2+-binding site is produced in the presence of E2. The second site is apparently altered by increasing the ionic strength. It is proposed that the second site may be at the interface between the phosphatase and E2, with Ca2+ acting as a bridging ligand for specific attachment of the phosphatase to E2.     

Investigation of the forms of pig duodenal Ca2+-binding protein produced by limited tryptic hydrolysis.

Vitamin D-dependent Ca2+-binding protein from pig duodenum was hydrolysed with trypsin in the presence of Ca2+ and two products were obtained: T1, which differed from the native protein by loss of Ac-Ser-Ala-Gln-Lys from the N-terminus and Ile-Ser-Gln-OH from the C-terminus, and T2, which differed from T1 by loss of a C-terminal lysine. The hydrolysis inactivated one of the two high-affinity Ca2+-binding sites on the native protein, and the remaining site was stable in T1 but labile in T2 when the proteins were Ca2+-free. Binding studies showed that T1 had Kd values of 2.8 +/- 0.1 nM, 57 +/- 13 microM and 0.8 +/- 0.3 microM for Ca2+, Mg2+ and Mn2+ respectively, and T2 had Kd 2.2 +/- 0.3 nM for Ca2+. The affinity for Mn2+, together with the other Kd values, identified the site on T1 as the site on the native protein previously found to have Kd 0.6 microM for Mn2+, rather than one with Kd 50 microM for Mn2+. In contrast with both the native protein and another form of the protein with a single Ca2+-binding site, the intrinsic fluorescence of T1 and T2 was little affected by the addition of Ca2+. It was concluded that the active binding site in T1 and T2, and also the site in the native protein with the higher affinity for Mn2+, was probably in the C-terminal half of the molecule.     

Different conformation changes induced by calcium and terbium of the porcine intestinal calcium-binding protein

The fluorescence of 1-anilino-8-naphthalenesulfonate (ANS) is progressively enhanced with increasing concentration of it, showing a proportionate blue shift of the emission maximum, by the interaction with the porcine intestinal Ca2+-binding protein (CaBP) in the absence of Ca2+. The apo-CaBP has a single binding site for ANS as determined by the fluorescence change, the apparent dissociation constant (Kd) estimated at 49.1 microM. Addition of Ca2+ or Tb3+ to the ANS-apo-CaBP system is capable of enhancing its fluorescence up to about 2- or 5-fold, respectively, causing further blue shift of the emission maximum. These metal ions do not affect the capacity of ANS binding, but Ca2+ slightly increases the Kd value. Increase of the fluorescence of the ANS-CaBP complex by increasing binding of Ca2+ to it was monophasic, while that with Tb3+ was biphasic, both saturated at the same molar ratio, 2, of added cations to the complex. Biphasic change of response has also been observed in UV absorption of the CaBP with increasing concentration of Tb3+. With a half-saturating concentration of Tb3+, Ca2+ can induce a much higher enhancement of the ANS fluorescence than excess Ca2+ alone. All these results indicate that the CaBP molecule contains a single ANS binding site and the conformation and/or microenvironment surrounding bound ANS of the protein is altered reversibly with binding of Ca2+ or Tb3+ to it and that there are differences between Ca2+- and Tb3+-induced conformation changes around the ANS-binding site and the tyrosine residue of it.     

One- and two-electron reduction of quinones by glutathione reductase

Yeast glutathione reductase (E.C. 1.6.4.2) catalyzes the oxidation of NADPH by p-quinones and ferricyanide with a maximal turnover number (TNmax) of 4-5 s-1.NADP+ stimulates the reaction and the TNmax/Km value of acceptors is reached at NADP+/NADPH greater than or equal to 100. TNmax is increased up to 30-33 s-1. The stimulatory effect of NADP+ may be associated with its complexation with the NADPH-binding site in the reduced enzyme (Kd = 40-60 microM). It is suggested that NADP+ shifts the electron density towards FAD in the two-electron-reduced enzyme and, evidently, changes its one-electron-reduction potentials, while quinones oxidize an equilibrium form of glutathione reductase containing reduced FAD. In the absence of NADP+ the reduction of quinones by glutathione reductase proceeds mainly in a two-electron manner. At NADP+/NADPH = 100 a one-electron reduction makes up 44% of the total process. At pH 6.0-7.0 the reduced forms of naphthoquinones undergo cyclic redox conversions. A hyperbolic dependence exists of the log TN/Km of quinones on their one-electron-reduction potentials.     

Quaternary structure of pigeon liver malic enzyme

Pigeon liver malic enzyme (EC 1.1.1.40) has a double dimer quaternary structure. The NADP+ analogs, aminopyridine adenine dinucleotide phosphate and nicotinamide-1,N6-ethenoadenosine dinucleotide phosphate, bind to the enzyme anti-cooperatively. In the presence of non-cooperative competing ligand NADP+, the binding parameter Hill coefficients of these analogues changed very little. Binding of L-malate with enzyme-AADP+ complex first enhanced then reduced the nucleotide fluorescence. Two L-malate binding sites, with Kd values of 23-30 and 270-400 microM, respectively. for the tight and weak binding sites were postulated. A hybrid model between the sequential and pre-existing asymmetrical models was proposed for the pigeon liver malic enzyme.     

Structural changes in the recombinant, NADP(H)-binding component of proton translocating transhydrogenase revealed by NMR spectroscopy

We have analysed 1H, 15N-HSQC spectra of the recombinant, NADP(H)-binding component of transhydrogenase in the context of the emerging three dimensional structure of the protein. Chemical shift perturbations of amino acid residues following replacement of NADP+ with NADPH were observed in both the adenosine and nicotinamide parts of the dinucleotide binding site and in a region which straddles the protein. These observations reflect the structural changes resulting from hydride transfer. The interactions between the recombinant, NADP(H)-binding component and its partner, NAD(H)-binding protein, are complicated. Helix B of the recombinant, NADP(H)-binding component may play an important role in the binding process.     

A K(+)-competitive fluorescent inhibitor of the H,K-ATPase.

The interactions of a novel fluorescent compound, 1-(2-methylphenyl)-4-methylamino-6-methyl-2,3-dihydropyrrolo[3,2-c ]quinoline (MDPQ) with the gastric H,K-ATPase were determined. MDPQ was shown to inhibit the H,K-ATPase and its associated K(+)-phosphatase competitively with K+, with Ki values of 0.22 and 0.65 microM, respectively. It also inhibited H+ transport with an IC50 of 0.29 microM, but at a concentration of 3.5 microM, reduced the steady-state level of phosphoenzyme by only 28%. The fluorescence of the inhibitor increased upon binding to the enzyme. 70% of this increment was quenched by K+, independently of Mg2+. The binding of MgATP to a high affinity site (K0.5(ATP) less than 1 microM) markedly increased the fluorescence due to the formation of an inhibitor-phosphoenzyme complex saturating with a K0.5(MDPQ) of 0.94 microM. The K(+)-dependent fluorescent quench (K0.5(K+) = 1.8 mM) required the ionophore, nigericin, indicating that K+ and MDPQ were competing at an extracytosolic site on the enzyme. Formation also of an enzyme-vanadyl-inhibitor complex was shown by the fact that Mg2+ plus vanadate enhanced MDPQ fluorescence in the absence of MgATP and decreased fluorescence in the presence of MgATP. The minimal stoichiometry of bound MDPQ determined by fluorescence titrations in the presence of MgATP was 1.4 mol/mol phosphoenzyme. The data suggest that this compound can serve as a probe of conformation at an extracytosolic site of the H,K-ATPase.     

Selective binding of cholesterol by recombinant fatty acid binding proteins

The sterol binding specificity of rat recombinant liver fatty acid binding protein (L-FABP) and intestinal fatty acid binding protein (I-FABP) was characterized with [3H]cholesterol and a fluorescent sterol analog dehydroergosterol. Ligand binding analysis, fluorescence spectroscopy, and activation of microsomal acyl-CoA:cholesterol acyltransferase activity showed that L-FABP-bound sterols. 1) Lipidex-1000 assay showed a dissociation constant Kd = 0.78 +/- 0.18 microM and stoichiometry of 0.47 +/- 0.16 mol/mol for [3H]cholesterol binding to L-PABP. 2) With [3H]cholesterol/phosphatidylcholine liposomes, the cholesterol binding parameters for L-FABP were Kd = 1.53 +/- 0.28 microM and stoichiometry 0.83 +/- 0.07 mol/mol. 3) L-FABP interaction with dehydroergosterol altered the fluorescence intensity and polarization of dehydroergosterol. Dehydroergosterol bound to L-FABP with Kd = 0.37 microM and a stoichiometry of 0.83 mol/mol. 4) Cholesterol and dehydroergosterol decreased L-FABP tyrosine lifetime. Dehydroergosterol binding produced sensitized emission of bound dehydroergosterol with longer lifetime.5) L-FABP bound two cis-parinaric acid molecules/molecule of protein. Cholesterol displaced one of these bound cis-parinaric acids. 6) L-FABP enhanced acyl-CoA:cholesterol acyltransferase in a concentration-dependent manner. In contrast, these assays indicated that I-FABP did not bind sterols. Thus, L-FABP appears able to bind 1 mol of cholesterol/mol of L-FABP, the L-FABP sterol binding site is equivalent to one of the two fatty acid binding sites, and L-FABP stimulates acyl-CoA:cholesterol acyltransferase by transfer of cholesterol.     

The interaction of Tb3+ with the human platelet surface

The interaction of the lanthanide Tb3+ with washed, human platelets was examined. When bound to the platelet surface, the fluorescence of this Ca2+ analog was increased approximately 200-fold, most likely by a F?rster mechanism involving platelet surface protein aromatic residues. The binding of Tb3+ to the unactivated platelet was specific and saturable with an apparent approximate Kd of 195 microM. Both Ca2+ and La3+ effectively displaced Tb3+ from platelet surface sites, but neither cation did so completely. Plasmin treatment of the platelet surface reduced Tb3+ fluorescence by 68% at saturation without significantly affecting the approximate apparent Kd. Activating washed, aspirinated platelets with ADP induced a 78% increase in Tb3+ fluorescence at saturation. Tb3+ competed effectively and completely for platelet surface-bound 45Ca2+ with an approximate IC50 of 10 microM. These data indicate the potential utility of this fluorescent lanthanide in characterizing Ca2+-binding sites on the human platelet.     

Investigation of the binding of Ca2+, Mg2+, Mn2+ and K+ to the vitamin D-dependent Ca2+-binding protein from pig duodenum.

The cation-binding properties of the vitamin D-dependent Ca2+-binding protein from pig duodenum were investigated, mainly by flow dialysis. The protein bound two Ca2+ ions with high affinity, and Mg2+, Mn2+ and K+ were all bound competitively with Ca2+ at both sites. The sites were distinguished by their different affinities for Mn2+, the one with the higher affinity being designated A (Kd 0.61 +/- 0.02 microM) and the other B (Kd 50 +/- 6 microM). Competitive binding studies allied to fluorimetric titration with Mg2+ showed that site A bound Ca2+, Mg2+ and K+ with Kd values of 4.7 +/- 0.8 nM, 94 +/- 18 microM and 1.6 +/- 0.3 mM respectively, and site B bound the same three cations with Kd values of 6.3 +/- 1.8 nM, 127 +/- 38 microM and 2.1 +/- 0.6 mM. For the binding of these cations, therefore, there was no significant difference between the two sites. In the presence of 1 mM-Mg2+ and 150 mM-K+, both sites bound Ca2+ with an apparent Kd of 0.5 microM. The cation-binding properties were discussed relative to those of parvalbumin, troponin C and the vitamin D-dependent Ca2+-binding protein from chick duodenum.     

Evidence of a calcium-induced structural change in the ATP-binding site of the sarcoplasmic-reticulum Ca2+-ATPase using terbium formycin triphosphate as an analogue of Mg-ATP

Terbium ions and terbium formycin triphosphate have been used to investigate the interactions between the cation and nucleotide binding sites of the sarcoplasmic reticulum Ca2+-ATPase. Three classes of Tb3+-binding sites have been found: a first class of low-affinity (Kd = 10 microM) corresponds to magnesium binding sites, located near a tryptophan residue of the protein; a second class of much higher affinity (less than 0.1 microM) corresponds to the calcium transport sites, their occupancy by terbium induces the E1 to E2 conformational change of the Ca2+-ATPase; a third class of sites is revealed by following the fluorescence transfer from formycin triphosphate (FTP) to terbium, evidencing that terbium ions can also bind into the nucleotide binding site at the same time as FTP. Substitution of H2O by D2O shows that Tb-FTP binding to the enzyme nucleotide site is associated with an important dehydration of the terbium ions associated with FTP. Two terbium ions, at least, bind to the Ca2+-ATPase in the close vicinity of FTP when this nucleotide is bound to the ATPase nucleotide site. Addition of calcium quenches the fluorescence signal of the terbium-FTP complex bound to the enzyme. Calcium concentration dependence shows that this effect is associated with the replacement of terbium by calcium in the transport sites, inducing the E2----E1 transconformation when calcium is bound. One interpretation of this fluorescence quenching is that the E1----E2 transition induces an important structural change in the nucleotide site. Another interpretation is that the high-affinity calcium sites are located very close to the Tb-FTP complex bound to the nucleotide site.     

Metal binding and metal-induced conformational change of frog bone gamma-carboxyglutamic acid-containing protein

Ca2+ or Cd2+ binding and the conformational change induced by the metal binding in two frog bone Gla-proteins (BGP, termed BGP-1 and BGP-2) were studied by equilibrium dialysis and CD measurement. By CD measurement in the far-ultraviolet region, the alpha-helix content of both apoBGPs was found to be 8%. Binding of both Ca2+ and Cd2+ was accompanied with a change in the CD spectrum, and the alpha-helix content increased to 15 and 25% for BGP-1 and BGP-2, respectively. CD measurement in the near-ultraviolet region indicated that the environment of aromatic amino acid residues in the protein molecule was changed by metal binding. Equilibrium dialysis experiments indicated that each of these two protein binds specifically 2 mol of Ca2+, and nonspecifically an additional 3-4 mol of Ca2+ in 0.02 M Tris-HCl/0.15 M NaCl (pH 7.4), at 4 degrees C. According to the two separate binding sites model, BGP-1 has 1 high-affinity Ca2+ binding site (Kd1 = 0.17 mM) and 1 low-affinity site (Kd2 = 0.29 mM), and BGP-2 contains 1 high-affinity site (Kd1 = 0.14 mM) and 1 low-affinity site (Kd2 = 0.67 mM). In addition, 2 Cd2+ bound to a high-affinity binding site on BGP-1 with Kd1 of 10.4 microM, and 1 Cd2+ bound to a low-affinity binding site with Kd2 of 41.5 microM. On the other hand, BGP-2 had three classes of binding sites and 1 Cd2+ bound to each binding site with Kd1 = 3.6 microM, Kd2 = 16.3 microM, Kd3 = 51.7 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)