Showing papers on "Cooperative binding published in 1973"

Binding of small molecules to proteins.

Abstract: The nuclear Overhauser effect (NOE) is defined as the change in integrated intensity of the nmr signal arising from one set of nuclei, when a second set is subjected to a saturating rf field. The theory and applications of this effect have been reviewed in a recent book by Noggle,' and its power in elucidating the geometry of small molecules has been convincingly demonstrated. In brief, the effect depends on the perturbations of populations of nuclear Zeeman states in the presence of the irradiating field. Taking as an example a two-spin system, the effects can be rationalized as follows. The Zeeman energy levels appropriate for two spin -?h nuclei of the same species are diagrammed in FIQURE 1. At thermal equilibrium the populations of the states are given by a Boltzmann distribution, with the /3/3 state having a slight excess population (p + 8) over the a@ and pa states, while the aa state is slightly less populated ( p 6) . Thermal equilibrium is established by the relaxation processes symbolized by the arrows, with transition probabilities W,,, W,, W,. Other things being equal, the intensities of the nmr transitions are proportional to the population differences between the upper and lower state connected by the transition. When a saturating rf field is applied (say at the proper frequency for nucleus 1 ), rapid transitions are excited between the levels j3/3 and aj3 equalizing their populations; the same occurs between levels /3a and aa. The populations eventually reach a new steady state. The new distribution is qualitatively determined by the relation between W, and W,,. If W2 is much more effective than W,, then the states aa and Bj3 can reach a population distribution near that in the unperturbed states; state /3a is thus less populated than at thermal equilibrium, while state a/3 is more populated. The observed nmr transitions of the second nucleus &3 + pa and a/3 + aa thus increase in intensity. This is the case usually observed, and has formed the basis of much of the application of the homonuclear Overhauser effect. We have recently observed, however, that in macromolecules a different relationship occurs that leads to new and interesting applications.:' The transition probabilities W, and W, are proportional to T,./ (1 + 4 0 ~ 7 ~ ~ ) and to T~~ respectively. For low molecular

Studies of glutamate dehydrogenase. The influence of ADP, GTP, and L-glutamate on the binding of the reduced coenzyme to beef-liver glutamate dehydrogenase.

Abstract: The detailed investigation of the binding of NAD(P)H to beef liver glutamate dehydrogenase in the presence of the effectors ADP and GTP shows that each oligomer (composed of six identical polypeptide chains) has a total of I8 nucleotide binding sites. They can be subdivided into three classes. A first set of six binding sites, the active sites, is occupied by NAD(P)H with nearly the same affinity for both coenzymes, and accompanied by negative cooperativity between the binding sites within the oligomer. The second set of six binding sites, the nonactive sites, binds the coenzyme less tightly than the first set and ADP competes with NAD(P)H for these sites. The third set of six binding sites, the GTP sites, is responsible for the GTP binding. ADP abolishes the negative cooperativity of the NAD(P)H binding to the active sites and causes a weaker binding of both reduced coenzymes to these sites (KE. ADP, NAD(P)H = 70 pM for NADH and 64 pM for NADPH). Also the reduced coenzymes weaken the binding of ADP to the enzyme. The dissociation constant for the enzyme * ADP complex is found to be 17 pM in the presence and 3 pM in the absence of NAD(P)H. The results are interpreted on the basis of free energy conservation in internal equilibria. The coupling energy AE”, was calculated to be about I kcal/mol. In contrast to ADP/NAD(P)H, GTP and NAD(P)H enhance each other’s binding to glutamate dehydrogenase causing positive cooperativity which is stronger in the case of NADH than of NADPH. The Hill coefficient is found to be 2.1 for NADH and 1.8 for NADPH. The nonactive sites are occupied by NADH in the presence of GTP with a dissociation constant of 23 $5 indicating a three-fold enhancement of the affinity of the enzyme for NADH in the presence of GTP. This is not the case with NADPH, where the dissociation constant is about 600 pM. This value is similar to the dissociation constant of the binary complex. The influence of glutamate on the enzyme * NAD(P)H binding is similar to the effect obtained in the presence of GTP; glutamate and GTP, however, are most likely bound to Merent sites. Glutamate induces strong positive cooperativity of three of the active sites within the oligomer characterized by a Hill coefficient of 2.4 for both reduced coenzymes. The extrapolations yield dissociation constants of 2-3 pM for both reduced coenzymes. The nonactive sites are occupied with dissociation constants of 57 pM for NADH and 700 pM for NADPH. These data indicate that glutamate has only a small effect on the binding of the reduced coenzyme to the nonactive site.

The Cooperativity of γ-Aminobutyric Acid Action on the Membrane of Locust Muscle Fibers

Abstract: Dose-response curves of the action of γ-aminobutyric acid to increase the conductance of postsynaptic membrane were measured in fibers of the flexor tibialis muscle of the metathoracic leg of Locusta migratoria . The sigmoid shape of the curves is interpreted in terms of the cooperative binding of more than 1 γ-aminobutyric acid molecule to the receptor. The "function of state," which describes the particular case in which receptor activation is induced only after the binding of more than 1 ligand molecule, is compared with some other formulations of cooperative binding, and is shown to give a better fit to the experimental data. This function uniquely predicts that the limiting Hill slope at small concentrations equals n , the number of molecules required to activate a receptor. In the present experiments n appears to be 3.

Interaction of the polyelectrolyte heparin with copper(II) and calcium.

Abstract: The binding of copper(II) ions by heparin was investigated using equilibrium dialysi techniques, and the effects of this binding on solution properties determined In neus tral Tris buffer solutions, heparin binds a maximum of twenty-three to twenty-four copper ions in two classes of sites, one containing three to four binding sites, the other containing twenty to twenty-one sites Cooperative binding is associated with the larger class of sites In more acidic citrate buffer solution, only one class of sites is observed, containing about four to five binding sites Association constants are calculated for the classes and the possible chemical nature of the sites is discussed The binding of calcium ions in neutral buffer is also examined, and these ions appear to be bound by a group of twenty to twenty-one binding sites, with a larger association constant than that for the copper ions Definite effects on the solution properties of heparin, such as intrinsic viscosity, sedimentation coefficients, and partial specific volume, can be observed only in the cooperative binding of copper ions in neutral buffer The interpretation of these solution properties in terms of molecular size and shape is analyzed, and it is concluded that the metal ion interactions cause no major change in the apparently random coil conformation of heparin in buffered solution, although some minor changes can be associated with the cooperative uptake of copper ions

Activation of a Covalent Enzyme-Substrate Bond by Noncovalent Interaction with an Effector

Abstract: The absorption spectrum of an activesite specific chromophoric acyl enzyme, sturgeon β-(2-furyl)-acryloyl-glyceraldehyde-3-phosphate dehydrogenase, is reported. This acyl enzyme undergoes all of the catalyzed reactions characteristic of the intermediate of the physiological acyl enzyme, 3-phospho-D-glyceroyl-glyceraldehyde-3-phosphate dehydrogenease. The rates of reactions of both these acyl enzymes depend strongly on the extent of interaction of the acyl enzyme with the oxidized coenzyme, NAD+, even where the “redox” properties of the coenzyme are not required. Likewise, the spectral properties of chromophoric acyl enzyme are affected by the extent of bound NAD. Under the pseudophysiological conditions reported herein, there is a stoichiometric limitation of two furylacryloyl-acyl groups per enzyme molecule containing four covalently-equivalent subunits. The binding of NAD both to the apoenzyme and to the diacyl enzyme is heterogeneous: at low extents of NAD occupancy, NAD binding is stronger. The binding to acyl enzyme can be quantitatively described by an enzyme model involving a tetramer with 2-fold symmetry, and consequently containing equal numbers of two classes of sites. NAD binding to difurylacryloyl-enzyme occurs virtually discretely, first to the two unmodified (tight-binding) sites, followed by looser binding to the two acyl-sites. NAD occupancy at these latter sites transforms the chromophoric acyl spectrum from that characteristic of a model furylacryloyl-thiol ester in H2O to a highly perturbed furylacryloyl spectrum characteristic of monomeric native “active-thiol” furylacryloyl-enzymes. Likewise the acyl reactivity towards arsenolysis depends on the extent of NAD bound to the loose sites. Elimination of the tight binding of NAD to the difurylacryloyl enzyme tetramer by alkylation of the remaining two free SH groups with iodoacetate has no apparent influence on the NAD-dependent furylacryloyl-spectral perturbation at the “two equivalent acyl sites,” even though it eliminates the apparent “negative cooperativity” in NAD binding.

Negatively co-operative ligand binding

Abstract: Simple systems are considered in which the binding of a ligand at a single site exhibits a doubly sigmoid curve when saturation is plotted against the logarithm of ligand concentration, i.e. where a fraction of the site exhibits one dissociation constant and the rest exhibits another. The condition for this to occur is that the ligand should also combine at another site on the binding molecule with comparable affinity and that the binding at one site should markedly lower affinity at the other. The protonation of simple compounds such as cysteine and 3-hydroxypyridine is taken as an example of this process and the equations derived are also applied to the binding of substrates to enzymes.

Kinetic properties of phenylalanyl-tRNA and seryl-tRNA synthetases for normal substrates and fluorescent analogs.

Abstract: The kinetics of phenylalanyl-tRNA and seryl-tRNA formation were investigated with tRNAs and aminoacyl-tRNA synthetases from yeast. Phenylalanyl-tRNA synthetase yielded linear Lineweaver-Burk plots with tRNAphe, phenylalanine, and 1,N6-ethenoadenosine triphosphate (ɛATP) as variable substrates. According to equilibrium dialysis in the absence or presence of phenylalaninyl adenosine 5′-phosphate, phenylalanyl-tRNA synthetase possesses one binding site for phenylalanine. For ATP as variable substrate, the deviation from linearity in the Lineweaver-Burk plot, observed by other investigators, was confirmed. The slope of the curve indicates the presence of more than two ATP binding sites. Seryl-tRNA synthetase yielded a linear Lineweaver-Burk plot only with ɛATP as variable substrate. The Lineweaver-Burk plots for serine and tRNASer were non-linear; the interpretation we favor involves positive cooperativity between amino acid binding sites and between tRNA binding sites. Hill plots of the kinetic data showed that the enzyme possesses at least two binding sites for each of these substrates. The kinetic data for ATP could be interpreted as showing more than two binding sites with negative and positive cooperativity in binding of successive ATP molecules. The aminoalkyl adenylates, phenylalaninyl adenosine 5′-phosphate and serinyl adenosine 5′-phosphate, competitively inhibited the aminoacylation reaction with respect to amino acid. ɛATP functions in place of ATP in phenylalanyl-tRNA and seryl-tRNA formation although with rather different kinetic properties. Modified tRNAphe and tRNASer, in which the 3′-terminal adenosine was replaced by ethenoadenosine, were prepared by a C-C-A transferase-catalyzed reaction of ɛATP. These modified tRNAs show kinetic properties very similar to those of the unmodified tRNAs and can therefore be used, in place of the unmodified tRNAs, as fluorescent probes in synthetase-tRNA interaction studies. The kinetic behavior of phenylalanyl-tRNA synthetase appears to be much simpler than that of seryl-tRNA synthetase, despite the fact that the former enzyme is twice as big and contains twice as many subunits as the latter one. The comparative simplicity of the one enzyme relative to the other correlates with previous results on interactions with substrates, which were obtained by fluorescence measurements and nuclease protection studies.

Cooperative nonenzymic base recognition. Kinetics of the binding of a base monomer to a complementary polynucleotide template

Abstract: The kinetics of double-helix formation by poly U and the complementary monomer N-6,9-dimethyladenine (m6m9A) has been measured using a new fast temperature-jump apparatus. The cooperative binding kinetics are complicated by the extensive self-association of the monomers, but a satisfactory analysis using average relaxation times was possible in terms of three different models. Application of a model which considers only monomer binding yields the upper limit for the binding rate constant of an m6m9A monomer next to an already bound monomer on a poly U strand: (2 ± 0.4) × 108M−1sec−1. A lower limit is found by using a model which allows for binding of all m6m9A stacks to poly U with equal rate constants: (3 ± 0.3) × 107M−1sec−1. A third model with “weighted” rate constants consistent with the data: (7.5 ± 1.0) × 107M−1sec−1. The rate of cooperative binding of m6m9A to the trimer UpUpU has also been measured. The rate constants obtained with the trimer agree with those obtained with the polymer for each of the three models within experimental error.

Cooperative binding of oxygen to hemoglobin: analysis of accurate equilibrium binding data.

Abstract: Accurate equilibrium binding data for the oxygenation of hemoglobin are used (a) to show that various models for cooperativity are inconsistent with the best available experimental data, (b) to determine the equilibrium constants for binding of 2,3-diphosphoglycerate to hemoglobin molecules in intermediate stages of oxygenation, and (c) to deduce a mechanism for allosteric effects in hemoglobin which is consistent with the best available experimental data. The total free energy of cooperativity is defined and discussed.

Tertiary and Quaternary Structural States of Hemoglobin

Abstract: X-ray diffraction studies have led to the solution of the structures of several conformational states of hemoglobin, including some partially oxygenated and partially oxidized forms. Comparison of these structures is beginning to show the conformational changes involved in the cooperative binding of oxygen to hemoglobin. This paper will attempt to summarize the data at hand and indicate where the missing structural information can be found.