Equilibrium and kinetic analysis of nucleotide binding to the DEAD-box RNA helicase DbpA

The Escherichia coli DEAD-box protein A (DbpA) is an RNA helicase that utilizes the energy from ATP binding and hydrolysis to facilitate structural rearrangements of rRNA. We have used the fluorescent nucleotide analogues, mantADP and mantATP, to measure the equilibrium binding affinity and kinetic mechanism of nucleotide binding to DbpA in the absence of RNA. Binding generates an enhancement in mant-nucleotide fluorescence and a corresponding reduction in intrinsic DbpA fluorescence, consistent with fluorescence resonance energy transfer (FRET) from DbpA tryptophan(s) to bound nucleotides. Fluorescent modification does not significantly interfere with the affinities and kinetics of nucleotide binding. Different energy transfer efficiencies between DbpA-mantATP and DbpA-mantADP complexes suggest that DbpA adopts nucleotide-dependent conformations. ADP binds (K(d) approximately 50 microM at 22 degrees C) 4-7 times more tightly than ATP (K(d) approximately 400 microM at 22 degrees C). Both nucleotides bind with relatively temperature-independent association rate constants (approximately 1-3 microM(-1) s(-1)) that are much lower than predicted for a diffusion-limited reaction. Differences in the binding affinities are dictated primarily by the dissociation rate constants. ADP binding occurs with a positive change in the heat capacity, presumably reflecting a nucleotide-induced conformational rearrangement of DbpA. At low temperatures (<22 degrees C), the binding free energies are dominated by favorable enthalpic and unfavorable entropic contributions. At physiological temperatures (>22 degrees C), ADP binding occurs with positive entropy changes. We favor a mechanism in which ADP binding increases the conformational flexibility and dynamics of DbpA.     

Investigations of spectrin-lipid interactions using fluoresceinphosphatidylethanolamine as a membrane probe

The binding of human erythrocyte spectrin to large unilamellar vesicles (LUVET) formed by the extrusion technique has been studied using fluoresceinphosphatidylethanolamine (FPE) as a reporter of electrostatic membrane potential. Spectrin aliquots were added to a suspension of FPE-labelled LUVETs to elucidate both the type of charge involved and the dissociation constants for spectrin binding to various lipids. All binding experiments showed serial increases in FPE fluorescence intensity upon serial additions of spectrin, indicative of increasing positive charge at the membrane surface. This proves for the first time that although exhibiting an overall net negative charge, spectrin binds to lipid surfaces by presenting positive charges to the lipid surface. Binding curves were obtained from the change in fluorescence intensity upon each spectrin addition and analysed to determine dissociation constants. A K(d) of 0.14+/-0.12 microM was found for spectrin binding to FPE-labelled phosphatidylcholine/phosphatidylserine (PC/PS) LUVETs at 22 degrees C in high salt conditions. A similar K(d) of 0.17+/-0.11 microM was obtained for spectrin binding to neutral LUVETs composed of PC. However, binding was found to be much weaker for PC/PS LUVETs under low salt conditions with a K(d) of 1.22+/-0.48 microM.     

Synthesis of thiol-reactive, long-wavelength fluorescent phenoxazine derivatives for biosensor applications

Two environmentally sensitive, long-wavelength fluorescent phenoxazine derivatives, INR and IANR, were synthesized with linkers for conjugation to the thiol group of cysteine in binding proteins. The linkers were designed based on the attachment sites at two different positions on the phenoxazine, which were chosen in order to study the orientation of the dye with respect to the binding protein. Conjugation of the dyes to the S337C maltose binding protein (MBP) mutant provided conjugates of these dyes that are capable of detecting maltose with different sensitivities. The dye INR gave a 3-fold (+200%) change in fluorescence intensity upon maltose binding when conjugated to S337C MBP with a binding constant (K(d)) of 435 microM. The fluorescence change for IANR was only 20% and the K(d) was 1.4 mM. Conformational analysis of the dyes by molecular modeling suggested that the linker in IANR imparted greater conformational freedom to the dye, resulting in little change in environment between the open and the closed-form conformations. The linker in INR, on the other hand, showed restricted motion, which placed the dye in different environments in the open and closed forms of the protein. Thus, design and placement of the linker play a critical role in the performance of these dyes as environmentally sensitive probes.     

Thermal stability and ligand-binding properties of human plasma alpha 1-acid glycoprotein (orosomucoid) as determined with fluorescent probes

The fluorescence of 1,8-anilinonaphthalene sulfonate is enhanced and blue-shifted upon binding to alpha 1-acid glycoprotein, a human plasma protein of uncertain function. Fluorescence titrations of delipidated protein indicate at least two classes of binding sites having dissociation constants of 0.33 microM and 12 microM at 25 degrees C in 0.02 M potassium phosphate/0.15 M NaCl, pH 7.4. Exclusion chromatography measurements indicate only 1 binding site per mol protein, suggesting that the heterogeneity is due to differences between protein molecules, the origin of which remains unclear. The fluorescence of a mixture of dye and protein is progressively diminished upon addition of ethanol and other organic solvents whose presence could be detected at concentrations as low as 100 mM. Addition of the adrenergic drug propranolol to a mixture of alpha 1-acid glycoprotein (2.5 microM) and 1,8-anilinonaphthalene sulfonate (4 microM) caused a hyperbolic decrease in dye fluorescence to 30% of the initial value, with half-maximal response near 1 microM propranolol. When the protein-dye mixture was heated, the fluorescence of the dye exhibited a reversible downward transition with midpoint near 65 degrees C, compared to a midpoint of 58.5 degrees C obtained by intrinsic fluorescence in the absence of dye. This stabilization was confirmed with fluorescein-labeled protein, whose fluorescence polarization revealed a melting transition at 58.8 degrees C in the absence of ligands which increased by 5-6 Cdeg in the presence of 1,8-anilinonaphthalene sulfonate or propranolol. The sensitivity of 1,8-anilinonaphthalene sulfonate fluorescence to changes in the conformation and ligand environment of alpha 1-acid glycoprotein should facilitate efforts to understand the structure and function of this acute-phase reactant.     

Fiber-optic immunosensor for mycotoxins

Evanescent wave-based fiber-optic immunosensors were studied for the detection of fumonisins and aflatoxins in maize. Two formats, competitive and non-competitive, were used. A competitive format was used to measure fumonisin B1 (FB1) in both spiked and naturally contaminated maize samples. Fumonisin monoclonal antibodies were covalently coupled to an optical fiber and the competition between FB1 and FB1 labeled with fluorescein (FB1-FITC) for the limited number of binding sites on the fiber was assessed. The signal generated in the assay was inversely proportional to the FB1 concentration. For samples, the concentration causing an inhibition of binding by 50% (IC50) was dependent upon the clean-up procedure used. Simple dilution of methanolic maize extracts yielded an assay with an IC50 equivalent to 25 microg FB1 g(-1) maize with a limit of detection of 3.2 microg g(-1) maize. Affinity column clean-up yielded an assay with an IC50 equivalent to 5 microg FB1 g(-1) maize (limit of detection 0.4 microg FB1 g(-1)). An HPLC method and the immunosensor method agreed well for naturally contaminated maize samples except when large amounts of other fumonisins that cross-react with the immunosensor were present. The second sensor format, for the mycotoxin aflatoxin B1 (AFB1), was a non-competitive assay using the native fluorescence of this mycotoxin. Because the fluorescence of AFB1 itself was detected, the response of the sensor was directly proportional to the toxin concentration. The sensor, while capable of detecting as little as 2 ng ml(-1) of AFB1 in solution was technically not an immunosensor, since the attachment of aflatoxin specific antibodies was not required. Sensors of the formats described have the potential to rapidly screen individual maize samples but require coupling with a clean-up technique to be truly effective.     

Binding of phlorizin to the isolated C-terminal extramembranous loop of the Na+/glucose cotransporter assessed by intrinsic tryptophan fluorescence

Phlorizin, a phloretin 2'-glucoside, is a potent inhibitor of the Na(+)/glucose cotransporter (SGLT1). On the basis of transport studies in intact cells, a binding site for phlorizin was suggested in the region between amino acids 604-610 of the C-terminal loop 13. To further investigate phlorizin binding titration experiments of the intrinsic Trp fluorescence of isolated wild-type loop 13 and two mutated loops (Y604K and G609K) were carried out. Phlorizin (135 microM) produced approximately 40% quenching of the fluorescence of wild-type loop 13; quenching could also be observed with the two mutated loops. The apparent K(d) was lowest for the wild-type loop 13 (K(d) approximately 23 microM), followed by mutant G609K (57 microM) and mutant Y604K (70 microM). Binding of phlorizin was further confirmed by a decrease of the accessibility of loop 13 to the collisional quencher acrylamide. The interaction involves the aromatic moiety of the aglucone since phloretin (the aglucone of phlorizin) showed almost the same effects as phlorizin, while d-glucose did not. MALDI-TOF experiments revealed that loop 13 contained a disulfide bond between Cys 560 and Cys 608 that is very important for phlorizin-dependent fluorescence quenching. These studies provide direct evidence that loop 13 is a site (important amino acids including 604-609) for the molecular interaction between SGLT1 and phlorizin. They confirm that the aglucone part of the glucoside is responsible for this interaction.     

Method dependence of apparent stoichiometry in the binding of salicylate ion to human serum albumin: a comparison between equilibrium dialysis and fluorescence titration.

The binding of salicylate ion to human serum albumin (HSA) was studied in 100 mM potassium phosphate buffer (pH 7.4, 25 degrees C), using equilibrium dialysis and fluorescence titration methods. The protein samples tested were (a) dialyzed human plasma and (b) a commercial preparation of HSA, essentially free of globulin and fatty acids. Independent of the analytical method used, Scatchard and nonlinear regression analyses of the data pointed to a single class of high-affinity salicylate binding sites. On the other hand, the binding parameters were found to be method dependent. K(d) ranged between 25 +/- 2.4 and 62 +/- 15 microM in equilibrium dialysis and between 10 +/- 1.3 and 40 +/- 3.0 microM in fluorescence titration. (The higher limits refer to plasma samples at high [HSA]). Following the same pattern, the apparent stoichiometry of binding (though independent of sample identity and concentration) was higher in equilibrium dialysis (n(app) = 3.2 +/- 0.10) than in fluorescence titration (n(app) 1.9 +/- 0.30). The difference between the two methods could be reconciled by invoking two distinct classes of binding sites (I and II), which had identical (or marginally different) K(d) values, while differing in the magnitude of the fluorescence signal (Deltaf) generated upon ligand binding (Deltaf, PL(I) = Deltaf(I); Deltaf, PL(II) = 0). Further, it was assumed that the state of occupation of class II sites affected the fluorescence efficiency of class I sites, such that Deltaf, PL(I,II) = betaDeltaf(I) (beta = interaction factor). A random binding scheme involving P, PL(I), PL(II), and PL(I,II) was formulated. The model adequately predicted the behavior of the system when monitored through the change in protein fluorescence: Taking K(d) = 25 microM and n(T) = 3, the interaction factor beta was found to be 0.62 +/- 0.10. It was concluded that the correct parameters for the binding of salicylate ion to HSA are K(d) = 25 +/- 2.4 microM and n(T) = 3.2 +/- 0.10, as indicated by equilibrium dialysis of purified HSA. Besides updating information relating to the salicylate binding potential of HSA, this study serves to illustrate a likely complication in the study of protein-ligand interactions by fluorometric methods.     

Purine nucleoside phosphorylase from Cellulomonas sp.: physicochemical properties and binding of substrates determined by ligand-dependent enhancement of enzyme intrinsic fluorescence,and by protective effects of ligands on thermal inactivation of the enzyme

Purine nucleoside phosphorylase (PNP) from Cellulomonas sp., homotrimeric in the crystalline state, is also a trimer in solution. Other features of the enzyme are typical for "low molecular mass" PNPs, except for its unusual stability at pH 11. Purine bases, alpha-D-ribose-1-phosphate (R1P) and phosphate enhance the intrinsic fluorescence of Cellulomonas PNP, and hence form binary complexes and induce conformational changes of the protein that alter the microenvironment of tryptophan residue(s). The effect due to guanine (Gua) binding is much higher than those caused by other ligands, suggesting that the enzyme preferentially binds a fluorescent, most probably rare tautomeric anionic form of Gua, further shown by comparison of emission properties of the PNP/Gua complex with that of Gua anion and its N-methyl derivatives. Guanosine (Guo) and inosine (Ino) at 100 microM concentration show little and no effect, respectively, on enzyme intrinsic fluorescence, but their protective effect against thermal inactivation of the enzyme points to their forming weak binary complexes with PNP. Binding of Gua, hypoxanthine (Hx) and R1P to the trimeric enzyme is described by one dissociation constant, K(d)=0.46 microM for Gua, 3.0 microM for Hx, and 60 microM for R1P. The binding stoichiometry for Gua (and probably Hx) is three ligand molecules per enzyme trimer. Effects of phosphate on the enzyme intrinsic fluorescence are due not only to binding, but also to an increase in ionic strength, as shown by titration with KCl. When corrected for effects of ionic strength, titration data with phosphate are most consistent with one dissociation constant, K(d)=270 microM, but existence of a very weak binding site with K(d)>50 mM could not be unequivocally ruled out. Binding of Gua to the PNP/phosphate binary complex is weaker (K(d)=1.7 microM) than to the free enzyme (K(d)=0.46 microM), suggesting that phosphate helps release the purine base in the catalytic process of phosphorolysis.The results indicate that nonlinear kinetic plots of initial velocity, typical for PNPs, including Cellulomonas PNP, are not, as generally assumed, due to cooperative interaction between monomers forming the trimer, but to a more complex kinetic mechanism than hitherto considered.     

New type of pullulanase from Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis.

A new type of pullulanase which mainly produced panose from pullulan was found in Bacillus stearothermophilus and purified. The enzyme can hydrolyze pullulan efficiently and only hydrolyzes a small amount of starch. When pullulan was used as a substrate, the main product was panose and small amounts of glucose and maltose were simultaneously produced. By using pTB522 as a vector plasmid, the enzyme gene was cloned and expressed in Bacillus subtilis. Since the enzyme from the recombinant plasmid carrier could convert pullulan into not only panose but also glucose and maltose, we concluded that these reactions were due to the single enzyme. The new pullulanase, with a molecular weight of 62,000, was fairly thermostable. The optimum temperature was 60 to 65 degrees C, and about 90% of the enzyme activity was retained even after treatment at 60 degrees C for 60 min. The optimum pH for the enzyme was 6.0.     

Binding of NAD+ to pertussis toxin.

The equilibrium dissociation constant of NAD+ and pertussis toxin was determined by equilibrium dialysis and by the quenching of the protein's intrinsic fluorescence on titration with NAD+. A binding constant, Kd, of 24 +/- 2 microM at 30 degrees C was obtained from equilibrium dialysis, consistent with the previously determined value for the Michaelis constant, Km, of 30 +/- 5 microM for NAD+ (when the toxin is catalysing the ADP-ribosylation of water and of dithiothreitol). The intrinsic fluorescence of pertussis toxin was quenched by up to 60% on titration with NAD+, and after correction for dilution and inner filter effects, a Kd value of 27 microM at 30 degrees C was obtained, agreeing well with that found by equilibrium dialysis. The binding constants were measured at a number of temperatures using both techniques, and from this the enthalpy of binding of NAD+ to toxin was determined to be 30 kJ.mol-1, a typical value for a protein-ligand interaction. There is one binding site for NAD+ per toxin molecule.     

Probing the nucleotide binding domain of the osmoregulator EnvZ using fluorescent nucleotide derivatives

EnvZ is a histidine protein kinase important for osmoregulation in bacteria. While structural data are available for this enzyme, the nucleotide binding pocket is not well characterized. The ATP binding domain (EnvZB) was expressed, and its ability to bind nucleotide derivatives was assessed using equilbrium and stopped-flow fluorescence spectroscopy. The fluorescence emission of the trinitrophenyl derivatives, TNP-ATP and TNP-ADP, increase upon binding to EnvZB. The fluorescence enhancements were quantitatively abolished in the presence of excess ADP, indicating that the fluorescent probes occupy the nucleotide binding pocket. Both TNP-ATP and TNP-ADP bind to EnvZB with high affinity (K(d) = 2-3 microM). The TNP moiety attached to the ribose ring does not impede access of the fluorescent nucleotide into the binding pocket. The association rate constant for TNP-ADP is 7 microM(-1) s(-1), a value consistent with those for natural nucleotides and the eucaryotic protein kinases. Using competition experiments, it was found that ATP and ADP bind 30- and 150-fold more poorly, respectively, than the corresponding TNP-derivatized forms. Surprisingly, the physiological metal Mg(2+) is not required for ADP binding and only enhances ATP affinity by 3-fold. Although portions of the nucleotide pocket are disordered, the recombinant enzyme is highly stable, unfolding only at temperatures in excess of 70 degrees C. The unusually high affinity of the TNP derivatives compared to the natural nucleotides suggests that hydrophobic substitutions on the ribose ring enforce an altered binding mode that may be exploited for drug design strategies.     

Direct measurement of the weak interactions between a mouse Fc receptor (Fc gamma RII) and IgG1 in the absence and presence of hapten: a total internal reflection fluorescence microscopy study.

Total internal reflection fluorescence microscopy (TIRFM) has been used to directly measure the weak dissociation constants of IgG with a mouse IgG receptor (moFc gamma RII) that has been purified and reconstituted into substrate-supported planar membranes. Dissociation constants were measured for three different mouse monoclonal anti-dinitrophenyl (DNP) IgG1 antibodies and for polyclonal mouse IgG, in the absence and presence of saturating amounts of hapten (DNP-glycine). The dissociation constant for polyclonal mouse IgG was 3 microM, which agrees well with previous results. The dissociation constants for the three monoclonal antibodies with moFc gamma RII ranged from 2 microM to 3 microM and were not statistically different, suggesting that changes in moFc gamma RII dissociation constants which may exist within the IgG1 subclass are less than the error of the TIRFM measurements (approximately 20%). The measured IgG1-moFc gamma RII dissociation constants were not different for individual monoclonal antibodies in the absence or presence of saturating concentrations of DNP-glycine, directly showing that possible allosteric changes which might occur upon hapten binding and affect the equilibrium characteristics of Fc receptor binding are small. This work demonstrates a new approach for quantitatively examining the effects of solution components on weak receptor-ligand interactions.     

Implications of the ligandin binding site on the binding of non-substrate ligands to Schistosoma japonicum-glutathione transferase

The binding interactions between dimeric glutathione transferase from Schistosoma japonicum (Sj26GST) and bromosulfophthalein (BS) or 8-anilino-1-naphthalene sulfonate (ANS) were characterised by fluorescence spectroscopy and isothermal titration calorimetry (ITC). Both ligands inhibit the enzymatic activity of Sj26GST in a non-competitive form. A stoichiometry of 1 molecule of ligand per mole of dimeric enzyme was obtained for the binding of these ligands. The affinity of BS is higher (K(d)=3.2 microM) than that for ANS (K(d)=195 microM). The thermodynamic parameters obtained by calorimetric titrations are pH-independent in the range of 5.5 to 7.5. The interaction process is enthalpically driven at all the studied temperatures. This enthalpic contribution is larger for the ANS anion than for BS. The strongly favourable enthalpic contribution for the binding of ANS to Sj26GST is compensated by a negative entropy change, due to enthalpy-entropy compensation. DeltaG degrees remains almost invariant over the temperature range studied. The free energy change for the binding of BS to Sj26GST is also favoured by entropic contributions at temperatures below 32 degrees C, thus indicating a strong hydrophobic interaction. Heat capacity change obtained for BS (DeltaC(p) degrees =(-580.3+/-54.2) cal x K(-1) mol(-1)) is twofold larger (in absolute value) than for ANS (DeltaC(p) degrees =(-294.8+/-15.8) cal x K(-1) mol(-1)). Taking together the thermodynamic parameters obtained for these inhibitors, it can be argued that the possible hydrophobic interactions in the binding of these inhibitors to L-site must be accompanied by other interactions whose contribution is enthalpic. Therefore, the non-substrate binding site (designed as ligandin) on Sj26GST may not be fully hydrophobic.     

Maltose-binding protein from the hyperthermophilic bacterium Thermotoga maritima: stability and binding properties

Recombinant maltose-binding protein from Thermotoga maritima (TmMBP) was expressed in Escherichia coli and purified to homogeneity, applying heat incubation of the crude extract at 75 degrees C. As taken from the spectral, physicochemical and binding properties, the recombinant protein is indistinguishable from the natural protein isolated from the periplasm of Thermotoga maritima. At neutral pH, TmMBP exhibits extremely high intrinsic stability with a thermal transition >105 degrees C. Guanidinium chloride-induced equilibrium unfolding transitions at varying temperatures result in a stability maximum at approximately 40 degrees C. At room temperature, the thermodynamic analysis of the highly cooperative unfolding equilibrium transition yields DeltaG(N-->U)=100(+/-5) kJ mol(-1 )for the free energy of stabilization. Compared to mesophilic MBP from E. coli as a reference, this value is increased by about 60 kJ mol(-1). At temperatures around the optimal growth temperature of T. maritima (t(opt) approximately 80 degrees C), the yield of refolding does not exceed 80 %; the residual 20 % are misfolded, as indicated by a decrease in stability as well as loss of the maltose-binding capacity. TmMBP is able to bind maltose, maltotriose and trehalose with dissociation constants in the nanomolar to micromolar range, combining the substrate specificities of the homologs from the mesophilic bacterium E. coli and the hyperthermophilic archaeon Thermococcus litoralis. Fluorescence quench experiments allowed the dissociation constants of ligand binding to be quantified. Binding of maltose was found to be endothermic and entropy-driven, with DeltaH(b)=+47 kJ mol(-1) and DeltaS(b)=+257 J mol(-1) K(-1). Extrapolation of the linear vant'Hoff plot to t(opt) resulted in K(d) approximately 0.3 microM. This result is in agreement with data reported for the MBPs from E. coli and T. litoralis at their respective optimum growth temperatures, corroborating the general observation that proteins under their specific physiological conditions are in corresponding states.     

The enzymatic reaction for the production of panose and isomaltose by glucosyltransferase from Aureobasidium

Glucosyltransferase from Aureobasidium produced 212 mg ml^-1 of glucosyl-oligosaccharides (panose: Glcα1→6Glcα1→4Glc 189 mg ml^-1 and isomaltose: Glcα1→6Glc 23 mg ml^-1) from maltose: Glcα1→4Glc at a high concentration (500 mg ml^-1) and the efficiency of production was 42-4%. The enzymatic reaction from maltose to panose is reversible but that from panose to isomaltose is not.     

Determination of the affinity of each component of a composite quaternary transition-state analogue complex of creatine kinase

Recombinant rabbit muscle creatine kinase (CK) was titrated with MgADP in 50 mM Bicine and 5 mM Mg(OAc)2, pH 8.3, at 30.0 degrees C by following a decrease in the protein's intrinsic fluorescence. In the presence of 50 mM NaOAc, but in the absence of added creatine or nitrate, MgADP has an apparent K(d) of 135 +/- 7 microM, and the total change in fluorescence on saturation (Delta%F) is 15.3 +/- 0.3%. Acetate was used as the anion in this experiment because it does not promote the formation of a CK.MgADP.anion.creatine transition-state analogue complex (TSAC) [Millner-White and Watts (1971) Biochem. J. 122, 727-740]. In the presence of 80 mM creatine, but no nitrate, the apparent K(d) for MgADP remains essentially unchanged at 132 +/- 10 microM, while Delta%F decreases slightly to 13.2 +/- 0.3%. In the presence of 10 mM nitrate, but no creatine, the apparent K(d) is once again essentially unchanged at 143 +/- 23 microM, but the Delta%F is markedly reduced to 4.2 +/- 0.2%. The presence of both 10 mM nitrate and 80 mM creatine during titration reduces the apparent K(d) for MgADP 10-fold to 13.7 +/- 0.7 microM, and Delta%F increases to 20.6 +/- 0.3%, strongly suggesting that the simultaneous presence of saturating levels of creatine and nitrate increases the affinity of CK for MgADP and promotes the formation of the enzyme*MgADP*nitrate*creatine TSAC. When the fluorescence of CK was titrated with MgADP in the presence of 80 mM creatine and fixed saturating concentrations of various anions, apparent K(d) values for MgADP of 132 +/- 10 microM, 25.2 +/- 1.3 microM, 18.8 +/- 0.9 microM, 13.7 +/- 0.7 microM, and 6.4 +/- 0.7 microM were observed as the anion was changed from acetate to formate to chloride to nitrate to nitrite, respectively. This is the same trend reported by Millner-White and Watts for the effectiveness of various monovalent anions in forming the CK.MgADP.anion.creatine TSAC. On titration of CK with MgADP in the presence of 80 mM creatine and various fixed concentrations of NaNO3, the apparent K(d) for MgADP decreases with increasing fixed concentrations of nitrate. A plot of the apparent K(d) for MgADP vs [NO3-] suggests a K(d) for nitrate from the TSAC of 0.39 +/- 0.07 mM. Similarly, titration with MgADP in the presence of 10 mM NaNO3 and various fixed concentrations of creatine gives a value of 0.9 +/- 0.4 mM for the dissociation of creatine from the TSAC. The data were used to calculate K(TDAC), the dissociation constant of the quaternary TSAC into its individual components, of 3 x 10(-10) M3. To our knowledge this is the first reported dissociation constant for a ternary or quaternary TSAC.     

Mn2+ binding to factor VIII subunits and its effect on cofactor activity

Metal ions, such as Ca2+ and Mn2+, are necessary for the generation of cofactor activity following reconstitution of factor VIII from its isolated light chain (LC) and heavy chain (HC). Titration of EDTA-treated factor VIII with Mn2+ showed saturable binding with high affinity (K(d) = 5.7 +/- 2.1 microM) as detected using a factor Xa generation assay. No significant competition between Ca2+ and Mn2+ for factor VIII binding (K(i) = 4.6 mM) was observed as measured by equilibrium dialysis using 20 microM Ca2+ and 8 microM factor VIII in the presence of 0-1 mM Mn2+. The intersubunit affinity measured by fluorescence energy transfer of an acrylodan-labeled LC (fluorescence donor) and fluorescein-labeled HC (fluorescence acceptor) in the presence of 20 mM Mn2+ (K(d) = 53.0 +/- 17.1 nM) was not significantly different from the affinity value previously obtained in the absence of metal ion (K(d) = 53.8 +/- 14.2 nM). The sensitization of phosphorescence of Tb3+ bound to factor VIII subunits was utilized to detect Mn2+ binding to the subunits. Mn2+ inhibited the phosphorescence of Tb3+ bound to HC and LC, as well as the HC-derived A1 and A2 subunits with a relatively wide range of estimated inhibition constant values (K(i) values = 169-1147 microM), whereas Ca2+ showed no effect on Tb3+ phosphorescence. These results suggest that factor VIII cofactor activity can be generated by Mn2+ binding to site(s) on factor VIII that are different from the high-affinity Ca2+ binding site. However, like Ca2+, Mn2+ did not alter the affinity for HC and LC association. Thus, Mn2+appears to generate factor VIII cofactor activity by a similar mechanism as observed for Ca2+following its association at nonidentical sites on the protein.     

Glucosyltransferase activity of commercial juice-processing enzyme preparations

Of various commercial enzyme preparations examined, Cytolase M102 was found to contain the highest glucosyltransferase activity (55 U ml⁻¹). It rapidly converted maltose to panose (Glcα1 → 6Glcα1 → 4Glc) with a V _max value of 5·8 mmol l⁻¹ min⁻¹ at 50°C in 0·05 mol l⁻¹ sodium acetate buffer (pH 4·4). The K _m value of the enzyme for maltose was 750 mmol l⁻¹. Yields of panose and glucose after 45 min of reaction, for example, were 47·2% and 52·8%, respectively, on the basis of the amount of maltose consumed.     

Intrinsic tryptophan fluorescence as a probe of metal and alpha-ketoglutarate binding to TfdA,a mononuclear non-heme iron dioxygenase

2,4-Dichlorophenoxyacetic acid (2,4-D)/alpha-ketoglutarate (alphaKG) dioxygenase, TfdA, couples the oxidative decarboxylation of alphaKG to the oxidation of the herbicide 2,4-D using a mononuclear non-heme Fe(II) active site. The intrinsic tryptophan fluorescence associated with the four Trp residues in TfdA allows for the use of fluorescence spectroscopy to monitor the binding of iron and alphaKG to the enzyme. The fluorescence spectrum of TfdA is quenched by 50-85% upon addition of Fe(II) or alphaKG, allowing determination of their binding affinities (K(d)=7.45+/-0.61 and 3.35+/-0.35 microM, respectively). Cu, Zn, Mn, Co, Mg, and Ca dictations also quench the TfdA fluorescence with affinities similar to that of Fe(II), whereas monovalent cations such as Na, K, and Li do not. H114A and D116A mutant forms of TfdA, lacking either a histidine or aspartate metallocenter ligand, exhibit weaker affinity for both Fe(II) and alphaKG based on the fluorescence changes. Trp256 is predicted to lie within 5 A of the metal and alphaKG binding sites; however, its substitution by Phe or Leu has negligible effects on the Fe(II)- and alphaKG-dependent fluorescence quenching. Because Trp195 is predicted to be quite distant ( approximately 15 A) from the active site, we conclude that some combination of Trp113 and Trp248 serves as the reporter that senses metal and cofactor binding to TfdA.