Rates Of Ligand Binding Research Articles

Dynamical transition of myoglobin revealed by inelastic neutron scattering.

Structural fluctuations in proteins on the picosecond timescale have been studied in considerable detail by theoretical methods such as molecular dynamics simulation, but there exist very few experimental data with which to test the conclusions. We have used the technique of inelastic neutron scattering to investigate atomic motion in hydrated myoglobin over the temperature range 4-350 K and on the molecular dynamics timescale 0.1-100 ps. At temperatures below 180 K myoglobin behaves as a harmonic solid, with essentially only vibrational motion. Above 180 K there is a striking dynamic transition arising from the excitation of nonvibrational motion, which we interpret as corresponding to torsional jumps between states of different energy, with a mean energy asymmetry of 12 kJ mol-1. This extra mobility is reflected in a strong temperature dependence of the mean-square atomic displacements, a phenomenon previously observed specifically for the heme iron by Mössbauer spectroscopy, but on a much slower timescale (10(-7) s). It also correlates with a glass-like transition in the hydration shell of myoglobin and with the temperature-dependence of ligand-binding rates at the heme iron, as monitored by flash photolysis. In contrast, the crystal structure of myoglobin determined down to 80 K shows no significant structural transition. The dynamical behaviour we find for myoglobin (and other globular proteins) suggests a coupling of fast local motions to slower collective motions, which is a characteristic feature of other dense glass-forming systems.

Effects of ligand size on pH and organic phosphate-dependent affinity changes in carp hemoglobin as measured by isonitrile binding

The equilibria of the binding of methyl and ethyl isonitrile to carp hemoglobin have been measured at three pH values in the presence and absence of inositol hexaphosphate. The binding of methyl isonitrile is characterized by a higher overall dissociation constant, C 1 2 , and a higher Hill coefficient, n, than that of the ethyl derivative. The former is consistent with the greater hydrophobicity of ethyl isonitrile, and the latter is probably due to a greater intrinsic difference or heterogeneity in the binding affinities of the α- and β-chains for the larger ligand. Changes in log C 1 2 which result from alterations in pH or addition of organic phosphate are the same for both ligands within experimental error. This result is not consistent with affinity changes being the result of steric interactions between the protein and the ligand. At pH 6 in the presence of inositol hexaphosphate, equilibrium parameters estimated from overall rates of ligand binding and dissociation are in good agreement with direct equilibrium measurements. This is consistent with the protein being in a low-affinity, T-like state even when saturated with ligand under these conditions, resulting in a loss of cooperativity in ligand binding. At high pH, ligand binding remains cooperative, as evidenced by n values greater than unity, a general lack of agreement between measured equilibrium parameters and those estimated from overall kinetic constants, and differences in the kinetics of ligand binding as observed by rapid-mixing and flash photolysis techniques. Thus, the deoxygenated state of carp hemoglobin at high pH does not appear to be a good model of a deoxygenated R quaternary structural state.

Regulation of the affinity state of the N-formylated peptide receptor of neutrophils: role of guanine nucleotide-binding proteins and the cytoskeleton.

Previous studies have indicated that the receptor for N-formylated peptides present on human neutrophils can exist in several ligand-dissociation states at least one of which is sensitive to guanine nucleotides. Human neutrophil membranes rich in cell surface enzyme markers have been isolated from cells pretreated at 37 degrees C with 5 nM fluoresceinated chemotactic peptide (N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein; Fl-peptide) or a buffer control and analyzed for receptor-ligand dissociation states using a previously published fluorescence assay for estimating ligand binding and dissociation rates (Sklar, L. A., et al. 1984. J. Biol. Chem. 259:5661-5669). Fractionation of crude microsomes derived from homogenates of unstimulated cells by ultracentrifugation on linear D2O gradients yielded two plasma membrane-rich fractions termed fast and slow microsomes. Analysis of Fl-peptide dissociation rates from receptor present in fast membrane fractions of unstimulated cells yielded data that could be best fit by assuming that the receptor exists in three distinct ligand-dissociation states. The intermediate ligand-dissociation state (state B) accounted for 47% of the total and was converted to the fastest ligand-dissociation state (state A) by incubation of membranes with GTP or GTP-gamma-S. The remainder of the receptor (17%) present in unstimulated membranes was in a state from which ligand was virtually nondissociable (state C). This form of the receptor was insensitive to GTP-gamma-S. When cells were stimulated with Fl-peptide, most of the receptor present in slow and fast membranes was of the state C type. In contrast to unstimulated cells, slow membranes derived from cells exposed to Fl-peptide contained the majority of the recoverable receptor indicating that receptor was transferred to a physically isolatable membrane domain after ligand binding to the intact cell. The ligand-induced formation of state C in both fast and slow microsome fractions was inhibited by treatment of cells with dihydrocytochalasin B. However, the drug had no effect on translocation of the receptor to slow membranes. Pertussis toxin treatment of intact cells had no effect on ligand-induced formation of state C in either fraction even though other cellular responses were inhibited. Both slow and fast membranes contained a 41-kD G protein as assayed by immunoblot analysis. The data suggest that ligand induces a segregation of receptor-ligand complexes into a membrane domain in which the receptor is functionally uncoupled from the 41-kD neutrophil G protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine nucleotide binding sites on beef heart F1-ATPase. Asymmetry and subunit location.

Previously we have shown that beef heart mitochondrial F1 contains a total of six adenine nucleotide binding sites. Three "catalytic" sites exchange bound ligand rapidly during hydrolysis of MgATP, whereas three "noncatalytic" sites do not. The noncatalytic sites behave asymmetrically in that a single site releases bound ligand upon precipitation of F1 with ammonium sulfate. In the present study, we find this same site to be the only noncatalytic site that undergoes rapid exchange of bound ligand when F1 is incubated in the presence of EDTA at pH 8.0. Following 1000 catalytic turnovers/F1, the site retains the unique capacity for EDTA-induced exchange, indicating that the asymmetric determinants are permanent and that the three noncatalytic sites on soluble F1 do not pass through equivalent states during catalysis. Measurements of the rate of ligand binding at the unique noncatalytic site show that uncomplexed nucleotide binds preferentially. At pH 7.5, in the presence of Mg2+, the rate constant for ADP binding is 9 X 10(3) M-1 s-1 and for dissociation is 4 X 10(-4) s-1 to give a Kd = 50 nM. The rate of dissociation is 10 times faster in the presence of EDTA or during MgATP hydrolysis, and it increases rapidly at pH below 7. EDTA-induced exchange is inhibited by Mg2+, Mn2+, Co2+, and Zn2+ but not by Ca2+ and is unaffected by dicyclohexylcarbodiimide modification. The unique noncatalytic site binds 2-azido-ADP. Photolysis results in the labeling of the beta subunit. Photolabeling of a single high-affinity catalytic site under conditions for uni-site catalysis also results in the labeling of beta, but a different pattern of labeled peptides is obtained in proteolytic digests. The results demonstrate the presence of two different nucleotide binding domains on the beta subunit of mitochondrial F1.

Rates of ligand binding to periplasmic proteins involved in bacterial transport and chemotaxis.

The ligand reactions of three binding proteins involved in bacterial transport and chemotaxis have been examined by stopped flow, rapid mixing techniques. The processes measured were: L-arabinose, D-galactose, and D-fucose binding to the Escherichia coli L-arabinose-binding protein; L-histidine binding to the Salmonella typhimurium L-histidine-binding protein; and D-maltose, maltotriose, cyclic maltohexaose, and cyclic maltoheptaose binding to the E. coli D-maltose-binding protein. Changes in tryptophan fluorescence were monitored, and the resultant time courses were analyzed quantitatively in terms of a simple one-step binding process. The fitted association rate constants for sugar binding are all about 1-3 X 10(7) M-1 s-1; variation in the affinity constants is expressed primarily by changes in the dissociation rate constants, 1-100 s-1. The sugar-binding proteins react at equal rates with the alpha and beta anomeric forms of their substrates. The ligand dissociation rates measured in vitro are consistent with the corresponding Vmax values observed for in vivo active transport. The association rate constant for the L-histidine-binding protein is 5-10 times greater than the corresponding rate constants for the sugar-binding proteins. A similar, large bimolecular rate, approximately 1 X 10(8) M-1 s-1, has been observed for the E. coli L-glutamine-binding protein (Weiner, J. H., and Heppel, L. A. (1971) J. Biol. Chem. 246, 6933-6941) and appears to reflect favorable electrostatic interactions between the charged amino acid and the surface of the protein molecule.

Electron transfer and ligand binding in metallo proteins

The multiple biological roles of metalloproteins may be illustrated by those macromolecules which are involved in the transport of dioxygen and its utilization for energy production. Reversible binding is the basic feature of oxygen carriers, such as hemoglobins or hemocyanins, and the stability of the complex as well as the dynamics of ligand binding to the metal(s) is modulated by the interactions with the proteins. Likewise, electron transfer between metalloproteins and/or the final electron acceptor, dioxygen, involves metal ions (Fe or Cu in most cases), but the specificity and rates of the processes in question are under protein control. The basic theme of this paper will be an illustration of the mechanisms of control exerted by the protein moiety on the intrinsic reactivity of the metal ions. It will be emphasized that the use of rapid reaction techniques is a very valuable approach to test the mechanism and to identify the intermediates involved in the control. For the sake of clarity, the regulatory action of the protein may be dealt with at a different level. At the tertiary level, the immediate environment of the metal ion controls the intrinsic rates of either ligand binding and/or electron transfer, as illustrated by kinetic experiments on myoglobin and azurin (taken as prototypes). In multisubunit proteins, the quaternary state of the macromolecule is involved in the control of reactivity. The classical allosteric model has been widely used to described cooperative effects in hemoglobin and hemocyanin; the general significance of the multiple quaternary state of a macromolecule will be illustrated by its applicability to cytochrome c oxidase, presenting a two-state kinetic model which accounts for some kinetic properties of this crucial enzyme.

Assignment of the quaternary structure of [formula omitted] hybrid hemoglobins: Implications for allosteric theories

The quaternary structures of α 2Znβ 2Fe(III), α 2Znβ 2Fe(III)N 3 −, α 2ZnβFe(II)CO, α 2Fe(III)β 2Zn, α 2Fe(III)N 3 −β 2Zn and α 2Fe(II)COβ 2Zn have been assigned using U.V. Circular dichroism, subunit dissociation measurements, and ligand binding rates. A “T” structure is obtained for all these Zn Fe hybrids. These results suggest that a complete R → T quaternary structure change does not occur until the third ligand binds. These observations, combined with previous studies of mixed valence Hb hybrids, place severe constraints on two state models of hemoglobin allostery.

The role of diffusion in limiting the rate of ligand binding to hemoglobin.

The Root effect hemoglobin from Thunnus thynnus, the Atlantic bluefin tuna, remains in the T state at acid pH even when liganded. There are two components in the binding of CO to the T state with rates of 0.01 and 1.0 X 10(6) M-1 S-1 at 20 degrees C, 0.1 M KPi, pH 6.0, and two components in binding to the R state with rates of 1.7 and 8.5 X 10(6) M-1 S-1 as measured by partial flash photolysis at 20 degrees C, 0.05 M Tris-HCl, pH 8.0. A single rate of NO binding of 1.8 X 10(7) M-1 S-1 is found at both pH 6 and pH 8. The rate of binding of NO to dimethyldeuterohemedisulfonate is much higher still at 5 X 10(8) M-1 S-1. The observed rate of NO binding to the protein is not affected by chain differences or by the R to T transition. It may measure the rate of diffusion of ligand to the vicinity of the heme site. The T state components have different NO dissociation velocity constants of 0.004 and 0.07 S-1. The apparent quantum yields for the T state components are approximately 0.035 and 0.0008; the high affinity component has the lower yield. A two-step scheme is used to explain the results. The presence of chain differences and allosteric states is reflected in the intrinsic binding rates of both NO and CO to the heme group.

Reduction and Subsequent Binding of Ruthenium Ions Catalyzed by Subcellular Components

The reduction of Cl(NH 3) 5Ru(III) and subsequent binding of heterocyclic ligands by the resultant (H 2O)(NH 3) 5Ru(II) ion is shown to be catalyzed by components of rat-liver cells. The presence of air significantly decreases the rate of heterocyclic ligand binding. In the case of microsome and soluble component catalysis, this is probably due to oxidation of the Ru(II) ion prior to complexation. Various inhibitors of electron-transfer proteins were employed in an effort to determine the preferred reducing species. These results lend support to the hypothesis that the antitumor activity of acido ruthenium(III) ammine complexes involves activation by reduction in vivo prior to metal coordination to nucleic acids. Anticancer drugs functioning by this mechanism may be preferentially toxic to or may localize in hypoxic areas of tumors.

Oxygen equilibria and ligand-binding kinetics of erythrocruorins from two burrowing polychaetes of different modes of life,Marphysa sanguinea andDiopatra cuprea

Oxygen equilibria, ligand-binding kinetics and some other physicochemical properties are reported for erythrocruorins of two intertidal polychaetes:Marphysa sanguinea, which inhabits simple, relatively stagnant burrows, andDiopatra cuprea, which inhabits impermeable, parchment-like tubes that are vigorously ventilated.Marphysa erythrocruorin has a higher O2 affinity, which is less pH dependent (at pH 7.3 and 20°C, half-saturation O2 tension,P50, and Bohr factor, ϕ=ΔlogP50/ΔpH, are 0.8 mm Hg and −0.25, respectively) than the corresponding parameters (P50=5.5; ϕ=−0.86) inDiopatra (Figs. 1 and 2). In contrast to vertebrate haemoglobins, inorganic salts increase erythrocruorin O2 affinity (Fig. 3). The kinetic rates of ligand binding and dissociation ofMarphysa andDiopatra erythrocruorins (Tables 1 and 2) correlate well with the measured O2 affinities and appear to illustrate basic molecular adaptations of the two species to their respective micro-environmental conditions.

On the mechanism of compound I formation from peroxidases and catalases

The present state of knowledge of the formation of the Compounds I of peroxidases and catalases is discussed in terms of the restrictions which must be placed upon a valid mechanism. It is likely that all Compounds I contain one oxygen atom bound to the heme-iron as in the Compound I of chloroperoxidase. Thus the formation of Compound I, obtained after molecular hydrogen peroxide and the enzyme diffuse together, involves a minimum of two bond ruptures and the formation of two new bonds. Yet this amazing reaction proceeds with an activation energy equal to or less than that for the fluidity of water. This result can only be accounted for by including at least one reversible step. Since Compound I formation requires the formation of an “inner sphere” complex, the presence or absence of water in the sixth co-ordination position of the heme-iron is of crucial importance. A comparison of the rates of ligand binding with the rate of Compound I formation indicate that the inner sphere complex leading to Compound I formation is formed by an excellent nucleophile, probably the peroxide anion, formed by a proton transfer from hydrogen peroxide. This proton cannot equilibrate with the bulk solvent. A proton derived from the active site would appear to be added to the hydroxide ion which permits a molecule of water to depart upon oxygen atom addition (or substitution) to (or at) the heme-iron. It is tentatively suggested that Compound I of catalase has a single active site per subunit molecule and that Compound I of peroxidase normally has two reactive sites.

Purification and properties of rat liver adenine phosphoribosyltransferase

The adenine phosphoribosyltransferase (AMP : pyrophosphate phosphoribosyltransferase, EC 2.4.2.7) of rat liver was purified to a specific activity of 1.1 μmol of AMP formed per min per mg. The enzyme activity is associated with an apparently homogeneous protein as shown by isoelectrofocusing, acrylamide gel electrophoresis, and N-terminal amino acid analysis (phenylalanine). The molecular weight of the enzyme was estimated to be approx. 20 000 by acrylamide gel electrophoresis in the presence of sodium dodecylsulfate and by sucrose density gradient zone sedimentation. The rat liver enzyme exhibited initial burst synthesis of AMP when 1-pyrophosphorylribose 5-phosphate was added. The 1-pyrophosphorylribose 5-phosphate initial-burst activity copurifies with the adenine phosphoribosyltransferase activity. A pH optimum of 10.0 was demonstrable for the adenine phosphoribosyltransferase. The initial-burst and steady-state phases of AMP synthesis catalyzed by highly purified rat liver adenine phosphoribosyltransferase have been partially characterized by the use of ligands which bind to sulfhydryl groups. Studies utilizing p-chloromercuribenzoate and HgCl 2 as inhibitors of AMP synthesis during the initial-burst and steady-state phases have revealed that sulfhydryl groups with different rates of ligand binding are present in the enzyme. The initial-burst phase was thereby delineated from the steady-state phase by use of these mercurial ligands. This delineation was also accomplished by titration with the Mg 2+ chelator, EDTA. The inhibitory effects of mercurials and EDTA were reversed by β-mercaptoethanol and excess Mg 2+, respectively. Quantitative binding studies with 5,5′-dithiobis(2-nitrobenzoic acid) and p-chloromercuribenzoate yielded values of 3.65 and 3.6 mol of sulfhydryl per mol of enzyme, respectively. 3.3 mol of cysteic acid per mol of performic acid-oxidized enzyme were found by amino acid analysis.

Studies on the mechanism of orthophosphate regulation of bovine brain hexokinase.

An attempt was made to gain insight into the mechanism of orthophosphate attenuation of glucose-6-P inhibition of bovine brain hexokinase I (ADP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from experiments of ligand binding and initial rate kinetics. Studies of glucose-6-P and phosphate binding to hexokinase reveal one binding site per hexokinase molecule. A model is presented which is consistent with the binding and kinetic data currently available on the alleviation of glucose-6-P inhibition of brain hexokinase by phosphate. The model implies that hexokinase may exist in equilibrium either as a free or phosphate-associated enzyme. The kinetic parameters of the two enzyme forms are similar except in their ability to bind glucose-6-P. It is suggested that the dissociation constant for glucose-6-P is relatively very high for hexokinase to which phosphate is bound. Phosphate appears to bind at an allosteric site on the enzyme, whereas glucose-6-P is associated either at the active site or at an allosteric site which overlaps the catalytic site.

Hemoglobin of the electric atlantic torpedo, Torpedo nobiliana: A cooperative hemoglobin without bohr effects

Hemoglobin of Torpedo nobiliana binds oxygen with a low degree of cooperativity, n ⩽ 1.5, but shows no appreciable pH dependence or response to organic phosphates. At neutral pH stripped Torpedo hemoglobin has a low oxygen affinity relative to stripped human hemoglobin with P 50 values of 15.8 mm Hg versus 0.85 mm Hg. The lower affinity of Torpedo hemoglobin can be attributed to slightly lower rates of ligand binding and considerably higher rates of ligand dissociation. Hemolysates of Torpedo hemoglobin show several electrophoretic zones, indicative of multiple hemoglobin components. Molecular weight studies indicate that Torpedo hemoglobin is normally tetrameric, but forms polymers readily under oxidizing conditions. In addition, Torpedo hemoglobin differs from most vertebrate hemoglobins in that NaCl increases its oxygen affinity. The NaCl effect is accompanied by increased rates of ligand binding.

Chain non-equivalence in nitric oxide binding to hemoglobin

The ratio of the apparent rates of ligand binding to the α and β subunits of human hemoglobin on mixing with non-saturating amounts of nitric oxide has been measured by two independent methods. Electron spin resonance measurements permit direct determination of the ratio of the amounts of the respective chains bound by NO. In stopped-flow kinetics measurements, use was made of the known difference in the kinetic constants of α and β chains in hemoglobin in the reaction with n-butyl isocyanide. Both methods concur in indicating that the apparent association rate constant of NO is greater for the α than for the β chain.

Effect of Carbon Dioxide on Human Hemoglobin

Abstract The kinetic basis for the reduction in the oxygen affinity of hemoglobin due to carbon dioxide at constant pH, is studied by measuring the deoxygenation rates of oxyhemoglobin with dithionite, as well as the rate of binding of carbon monoxide to deoxyhemoglobin. These studies were performed in buffer containing 0.05 m potassium phosphate, as well as various mixtures of potassium chloride and potassium bicarbonate all at a constant pH of 7.5 and 23°. The results indicate that the reduction in the ligand affinity of hemoglobin by carbon dioxide is the result of a reduced rate of ligand binding with no significant change in the rate of release. It is pointed out that this result is in contrast to salts which reduce the ligand affinity of hemoglobin by increasing the rate of ligand dissociation as well as reducing the rate of ligand binding.