Showing papers on "Cooperativity published in 1982"

Selective binding to polymers via covalent bonds. The construction of chiral cavities as specific receptor sites

Abstract: Separations of substances on polymers by fast and reversible covalent interactions are discussed. For this type of chemoselective affinity chromatography suitable new binding groups were developed for diols, monoalcohols and amines. The selectivity of the separations can be improved by cooperative binding of substrates via two or more binding groups. Polymers with binding groups located in a definite spatial proximity and cooperativity in cavities of specific shape show high selectivity for the resolution of racemates. These cavities can be looked at as models for natural receptor sites.

High-affinity binding and inhibition of calcium transport in a clonal cell line*

Abstract: and shows no cooperativity. The calcium antagonists inhibit potassium-induced 46Ca uptake into the cells with approximately the same potencies as those needed to inhibit [3H]nitrendipine binding to cell membranes. The affinity of these compounds for the PC12 cell calcium channel is slightly lower than that reported for binding to brain and heart. Potassium-stimulated 46Ca uptake into PC12 cells is rapid, being half-maximal within 30 s at 20 “C. Different classes of calcium antagonists seem to block calcium flux at different sites on the calcium channel. A lower limit of the rate of calcium movement through a single channel is given. PC12 cells seem to be a suitable model system for the study of the pharmacology and biochemistry of the voltage-dependent calcium channel.

Pigeon liver malic enzyme.

Abstract: Malic enzyme of pigeon liver is a tetrameric molecule with identical, or nearly-identical subunits. It catalyzes, in addition to oxidative decarboxylation of L-malate, the following metal activated component reactions: Oxalacetate decarboxylase; reductase with broad specificity on α-ketocarboxylic acids; a NADP+-dependent dismutation of L-malate to L-lactate; and proton exchange between pyruvate and medium water. The kinetic mechanism of oxidative decarboxylase is sequential and ordered, with NADP+ adding first to the metal enzyme, followed by L-malate, and by the release of products CO2, pyruvate, and NADPH. NADPH release, or a conformation change preceeding it, is rate-limiting in the overall reaction. Chemical modification studies indicate the presence of histidyl and lysyl residues at the nucleotide site, and tyrosyl residues at the carboxylic acid site. The involvement of protonated histidine(s) in NADPH binding is implicated by results of direct titration experiments, which also suggest a role of this residue as a proton sink in the catalytic reaction. A cysteinyl SH group is located near (but not at) each of the substrate-sites on the enzyme tetramer. Reaction of these groups with SH reagents causes selective loss of activities involving decarboxylation (i.e., oxidative decarboxylase, reductive carboxylase, and oxalacetate decarboxylase), owing to blockage of the reversible carbon-carbon cleavage step by the bulky substituent. All-of-the-sites reactivity is observed for non-specific thiol reagents such as 5,5′ dithiobis-(2-nitrobenzoic acid), N-ethylmaleimide, iodoacetate, and iodoacetamide. While bromopyruvate, which is reduced by the enzyme to L-bromolactate under catalytic conditions, alkylates these groups in an active-site directed manner with half-of-the-sites stoichiometry. The remaining two SH groups are reactive toward non-specific reagents, but at rates 2.4 - 3.6 fold lower than do the same groups on the unalkylated enzyme. This behavior is interpreted in terms of the ligand-induced negative cooperativity concept of Koshland, et al. (Biochemistry 5: 365–385, 1966): Reaction of bromopyruvate induces a conformation change on the alkylated subunit which is transmitted to the unoccupied subunit neighbor, turning off its catalytic site for reaction with L-malate, as well as converting the initial ‘fast’ SH groups into ‘slow’, or unreactive SH groups. In equilibrium binding experiments, all-of-the-sites reactivity is seen with nucleotide cofactors NADP+ and NADPH. Binding of Mn2+, or L-malate in the presence of Mn2+ and NADPH is biphasic, showing two ‘tight’ sites with dissociation constants in the micromolar range, and two ‘weak’ sites with 10–100 fold lower affinities. The presence of ‘tight’ and ‘weak’ L-malate sites is confirmed by fluorescence titration experiments which also yields similar affinities for the substrate molecule. In kinetic studies, two types of non-equivalent, and functionally distinct sites are detected. At saturating NADP+, and Mn2+ and L-malate levels corresponding to binding at tight sites, typical Michaelian behavior is observed. The reaction is inhibited uncompentitively by L-malate at higher concentrations corresponding to occupancy at all of the L-malate sites. Occupancy of Mn2+ at weak metal sites as well has no effect at low L-malate, but prevents substrate inhibition at high L-malate. A tentative ‘half-of-the-sites’ model consistent with results of chemical modification, binding, and kinetic experiments is proposed for this enzyme. This model implicates involvement of subunit cooperativity in the catalytic process. Malic enzyme is depicted as a tetramer composed of inititally identical subunits, each containing an active-site capable of binding all reactants. Mn2+ and L-malate bind anticooperatively to the tight and weak sites, in contrast to NADP+ which binds equivalently to all sites. On the fully active enzymes, only half (or the tight) of the subunits are simultaneously undergoing catalysis. Binding of L-malate (but not Mn2+) at the adjacent weak subunits causes a slow isomerization of the enzyme, and inhibition of NADPH dissociation from the catalytic subunits. Binding of Mn2+ at the same sites prevents this change and thereby relieving substrate inhibition. This model is further supported by results of active-site titration experiments, such as the half-size burst of enzyme-bound NADPH in the transient state, and half-of-the-sites reactivity of oxalate, an analog for the transition state intermediate of the reaction.

Evidence for hydrogen bonding of bound dioxygen to the distal histidine of oxycobalt myoglobin and haemoglobin

Abstract: The origin of the differences in oxygen binding energy in various haemoglobins and myoglobins has long been debated. Perutz1 proposed that the haem-coordinated histidine (proximal histidine) strains the haem iron in low affinity globins but relaxes it in high affinity globins. The existence of such tension in T-structure deoxyhaemoglobin (deoxyHb) was recently confirmed by electron paramagnetic resonance (EPR)2,3, resonance Raman4,5 and NMR6 spectroscopy. Although its contribution to the free energy of cooperativity is insignificant in the deoxy state, the tension at the haem is considered to be ∼1 kcal mol−1 for the ligated form in which the haem iron moves into the porphyrin plane7. The remaining free energy is probably stored in other parts of the molecule. Therefore, a study of the stabilization mechanisms of the oxygenated form became increasingly important. A hydrogen bond between the bound oxygen and the distal histidine has been proposed by Pauling8; this would be expected to stabilize the oxy form of the protein and could contribute to the regulation of the oxygen affinity through the oxygen dissociation rate. A series of EPR and functional studies on various cobalt-substituted monomeric haemoglobins and myoglobins suggested the presence of such hydrogen bonding8–12 and it has recently been established in crystals of oxy iron myoglobin (oxyFeMb)13 and in oxyhaemoglobin14. Here we present resonance Raman spectra of the oxy forms of cobalt–porphyrin-substituted myoglobin and haemoglobin (CoMb and CoHb) recorded in buffered H2O and D2O solutions at 406.7 nm excitation. Only the Raman lines corresponding to the O—O stretching mode of the bound oxygen15, appearing near 1,130 cm−1, are shifted (2–5 cm−1) on replacement of H2O by D2O; no other vibrations, including the Co—O2 stretching mode, exhibit any frequency shifts. This indicates that the bound oxygen in oxyCoMb and in both subunits of oxyCoHb interacts with the adjacent exchangeable proton, and confirms the formation of a hydrogen bond between the bound oxygen and the distal histidine9.

Probing the energetics of proteins through structural perturbation: sites of regulatory energy in human hemoglobin.

Abstract: The sites of energy transduction within the human hemoglobin molecule for the regulation of oxygen affinity have been determined by an extensive study of the molecule's energetic response to structural alteration at individual amino acid residues. For 22 mutant and chemically modified hemoglobins we have determined the total free energy used by the tetrameric molecule for alteration of oxygen affinity at the four binding steps. The results imply that the regulation of oxygen binding affinity is due to energy changes which are mostly localized at the alpha 1 beta 2 interface. They also indicate a high degree of "internal cooperativity" within this contact region--i.e., the structural perturbations at individual residue sites are energetically coupled. Cooperativity in ligand binding is thus a reflection of cooperativity at a deeper level--that of the protein-protein interactions within the alpha 1 beta 2 interfacial domain.

Quaternary-transformation-induced changes at the heme in deoxyhemoglobins.

Abstract: Quaternary-structure-induced differences in both the high- and low-frequency regions of the resonance Raman spectrum of the heme have been detected in a variety of hemoglobins. These differences may be the result of (1) changes in the amino acid sequence, induced by genetic and chemical modifications, and (2) alterations in the quaternary structure. For samples in solution in low ionic strength buffers, differences in the 1357-cm-1 line (an electron-density-sensitive vibrational mode) correlate with differences in the 216-cm-1 line (the iron-histidine stretching mode). Thus, changes in the iron-histidine bond and changes in the pi-electron density of the porphyrin depend upon a common heme-globin interaction. The quaternary-structure-induced changes in the vibrational modes associated with the heme demonstrate that there is extensive communication between the heme and the globin and impact on models for the energetics of cooperativity. The local interactions of the iron-histidine mode are energetically small and destabilize the deoxy heme in the T structure with respect to the R structure. Therefore, these interactions must be larger in the ligated protein than in the deoxy protein to obtain a negative free energy of cooperativity. Additionally, our data imply that the deprotonation of the proximal histidine does not play a major role in the energetics of cooperativity. On the other hand, models for cooperativity that require conformational changes in the iron-histidine bond or direct interaction between the porphyrin and the protein are qualitatively consistent with the observed variation of heme electronic structure in concert with protein quaternary structure.

Lipid-dependent membrane enzymes. A kinetic model for cooperative activation in the absence of cooperativity in lipid binding.

Abstract: The dependence of integral membrane enzymes on lipid activators in analyzed in terms of multiple binding site kinetics. Rate equations for an enzyme with n independent and indentical lipid binding sites are derived for the case that enzyme activity is proportional to the total amount of lipid bound, or that only fully substituted enzyme is active. A third equation applies to the case that lipids bind with infinite cooperativity to give fully substituted and active enzyme. None of the three models was entirely consistent with existing experimental data. The following kinetic model is shown to accommodate the degree of cooperativity observed in lipid activation experiments as well as the number of independent lipid-binding sites determined by electron-spin resonance measurements. The membrane enzyme is assumed to have n non-interacting and identical lipid-binding sites. Only fully substituted enzyme (ELn) and the next most highly substituted forms such as ELn-1 and ELn-2 may possess enzyme activity. These assumptions lead to cooperativity in activation. Cooperativity reaches a maximum when enzyme activity starts to appear with about 80% of the full lipid substitution. The increase in cooperativity is accompanied by a decrease in the lipid concentration required for half-maximal activation. Further kinetic aspects of a dynamic boundary lipid layer around integral membrane enzymes are discussed.

The effects of ethanol on the thermotropic properties of dipalmitoylphosphatidylcholine.

Abstract: The specific effect of ethanol on several aspects of the gel-to-liquid crystal transition of dipalmitoylphosphatidylcholine was investigated using two spectrophotometric techniques, one probe method and one direct method. Ethanol shifts the phase-transition temperature to low temperature, demonstrating that ethanol interacts preferentially with the fluid phase. Thermodynamic analysis of the melting point depression leads to a calculated membrane:buffer partition coefficient of 6.25 (mole fraction units) or 0.15 mole of ethanol per kilogram of lipid:mole of ethanol per liter of solution. Careful evaluation of the transition cooperativity with temperature resolution of +/- 0.1 degrees shows that there is no reduction in transition cooperativity, and thus no reduction in size of the cooperative lipid clusters due to ethanol. The implications of these findings for the mechanism of action of ethanol in terms of current theories of anesthetic mechanisms are discussed.

Reconstitution of the transferrin receptor in lipid vesicles. Effect of cholesterol on the binding of transferrin.

Abstract: Purified rabbit reticulocyte transferrin receptors were incorporated into phosphatidylcholine vesicles containing varying amounts of cholesterol. The binding of transferrin to the receptor in the reconstituted vesicles had three distinct characteristics: (1) The binding of transferrin exhibited the two components characteristic of transferrin binding to erythroid cells, a saturable, specific component and a nonsaturable, nonspecific component. (2) Transferrin binding exhibited positive cooperativity at low cholesterol/phospholipid (C/P) molar ratios. However, the cooperativity diminished and then disappeared as the C/P molar ratios were increased to the levels found in circulating red blood cells. (3) The amount of specific transferrin binding to the reconstituted vesicles also decreased as the C/P molar ratio was increased. These results indicate that in the reconstituted system the lipid environment plays a significant role in the expression of transferrin receptors.

Cooperative effects in α‐helices: An ab initio molecular‐orbital study

Abstract: Some properties of α-helices of polyclycine and polyalanine, up to the decapeptide, were investigated by ab initio molecular-orbital calculations. These helices were found to be unstable relative to the corresponding “fully extended chain” conformation. The electric field of helices of 8–10 residues is about 20% stronger than that of models built from noninteracting monomers, for example. This is a result of cooperativity, which is essentially governed by the intramolecular hydrogen bonds. The cooperativity is manifest in all properties of the helices: relative stability, dipole moment, proton affinity, electrical potential. The electric potential of helices of three and four residues is such that their instability can be compensated for by a single charged group acting as an “initiator.” The computed proton affinity of the (Ala)8 α-helix is about 45 kcal/mol larger than that of formamide, which confirms that long helices may be protonated at the carboxyl end in solution.

Interdependence of neurophysin self-association and neuropeptide hormone binding as expressed by quantitative affinity chromatography.

Abstract: The reciprocal modulation of neurophysin self-association and noncovalent peptide--protein interaction between neurophysin and the hormones oxytocin and vasopressin has been assessed by quantitative affinity chromatography. Competitive elutions of radiolabeled bovine neurophysin II (NPII) from the affinity matrices Met-Tyr-Phe-omega-(amino-hexyl)- [and (aminobutyl)-] agarose were performed with increasing concentrations of either of the soluble ligands oxytocin or lysine-vasopressin. Also, the dependence of NPII retardation by the same adsorbents on the concentration of applied protein was investigated in the absence of soluble ligand. The affinity constant of NPII for the immobilized peptide increased markedly with increasing amounts of applied protein and with the addition of small amounts of soluble ligand, the latter being more pronounced at higher protein concentrations. The affinity constant of the protein for the soluble ligand showed a smaller increase. The variation of l/(V - V0) (where V = the NPII elution volume and V0 = the elution volume of noninteracting control protein) with soluble ligand concentration was linear except near [ligand] = 0. The quantitative affinity chromatographic results on the tripeptidyl affinity columns are consistent with the view that NPII exists in a monomer in equilibrium dimer equilibrium, with the dimer exhibiting a stronger interaction with both neuropeptide and tripeptide analogues. The data also indicate that the self-associated protein dimer itself exhibits cooperativity, that is, stronger binding of the immobilized ligand at one site when a second site is occupied with a molecule of the soluble ligand than when no soluble ligand is bound. The deduction from the above of ligand-induced dimerization is evident also in the increased retardation of NPII on neurophysin--Sepharose when the eluting buffer contains soluble peptide hormone.

Activation of human erythrocyte Ca2+-dependent Mg2+-activated ATPase by calmodulin and calcium: quantitative analysis

Abstract: The effect of Ca2+ and calmodulin on (CaM) on the activation of Ca2+-dependent Mg2+-activated ATPase (Ca2+,Mg2+-ATPase; ATP phosphohydrolase, EC 3.6.1.3) has been carried out because of the finding that the CaM dependence of the activation varies with the concentration of free Ca2+, similarly to brain phosphodiesterase and adenylate cyclase. The study was carried out in the absence of chelating agents because they strongly interfere in the enzyme kinetics. Three main conclusions can be drawn (i) CaM-Ca3 and CaM-Ca4 together are the biochemically active species in vitro. (ii) These species bind in a non-cooperative way to the CaM-binding site of the enzyme with a dissociation constant of 6 x 10(-10) M or 1.1 x 10(-8) M, depending on whether Ca2+ saturates the substrate binding site of the enzyme or not. (iii) The binding of CaM-Ca3 to the enzyme lowers the dissociation constant of the enzyme for Ca2+ at the substrate binding site from 51.5 to 2.8 microM. Contrary to general belief, CaM does not induce pronounced positive cooperativity in the binding of Ca2+ to the enzyme. Such a cooperativity is seen only when the enzyme is incompletely saturated with the activator, but it disappears in the presence of saturating concentrations of CaM-Ca3. The rate equation proposed here accurately predicts the extent of enzyme activation over a wide range of Ca2+ and CaM concentration. In healthy erythrocytes the concentrations of Ca2+ and CaM are such that the Ca pump works with a minimal dissipation of energy, but a small increase in the intracellular Ca2+ concentration leads to a strong amplification of the pumping activity.

Ligation and quaternary structure induced changes in the heme pocket of hemoglobin: a transient resonance Raman study.

Abstract: The extent to which ligation and quaternary structure modify the heme-heme pocket configuration is determined by generating and analyzing transient resonance Raman spectra from various photolyzed and partially photolyzed hemoglobins (Hb). From small frequently shifts in Raman band I (approximately 1355 cm-1) it is determined that ligation induces a configurational change about the heme. The extent to which ligation modifies the heme pocket is influenced by the quaternary structure. With respect to the structural parameter responsible for variations in the pi orbital electron density of the porphyrin, the degree of alteration of the heme pocket configuration relative to deoxy-Hb(T) follows the sequence: liganded Hb(R) greater than liganded Hb(R) + IHP greater than liganded Hb(T) [alpha chain greater than beta chain] greater than deoxy-Hb(R). This progression of configurations also forms a sequence with respect to the "retentiveness" of the heme pocket as reflected in the ligand dynamics associated with geminate recombination. The results indicate that the heme-heme pocket of the R-state Hb's, relative to those of the T-state species, favors ligand retention in a dynamic, as well as thermodynamic, sense. The analysis of these and other related data implicates a ligation and quaternary structure modulated electronic and/or electrostatic interaction between the pi system of the porphyrin and the surrounding heme pocket as the basis for this variation in ligand dynamics as well as for the energetics of cooperativity.

Phosphorylation of calcium adenosinetriphosphatase by inorganic phosphate: reversible inhibition at high magnesium ion concentrations.

Abstract: Magnesium stimulates phosphorylation of the calcium pump protein of the sarcoplasmic reticulum by inorganic phosphate, but the effect is reversed by high [Mg2+]. This reversal is readily explained in terms of the generally accepted existence of two conformational states of the enzyme, E1 and E2. E2 is the form of the enzyme that can be phosphorylated by Pi, and it has one binding site for Mg2+. E1 is the form of the enzyme that has two high-affinity Ca2+ binding sites, and it is phosphorylated by ATP when Ca2+ is bound. Mg2+ can bind weakly to the two Ca2+ sites and to a third site known to be present on E1; this stabilizes E1 at the expense of E2 when [Mg2+] is large. Stabilization of E1 at pH 6.2 and 25 degrees C was found to be a highly cooperative function of [Mg2+] and was not prevented by increasing [Pi]. The latter result requires the existence of a binding site for Pi on E1, with an affinity for Pi comparable to that of E2. Cooperativity with respect to [Mg2+] requires that E2 is the stable state of the enzyme in the absence of ligands, with an equilibrium constant [E2]/[E1] on the order of 10(3) or higher at pH 6.2 and 25 degrees C.

Zn(II)-induced cooperativity of Escherichia coli ornithine transcarbamoylase

Abstract: The steady-state reaction of ornithine transcarbamoylase (ornithine carbamoyltransferase, carbamoyl phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) purified from the argI gene product of Escherichia coli strain K-12 exhibits Michaelis-Menten kinetics over an extended range of concentration for both L-ornithine and carbamoyl phosphate. In the presence of Zn2+, however, the saturation curve of L-ornithine becomes sigmoidal, revealing positive cooperativity for this anabolic enzyme. The kinetic data give a limiting Hill coefficient of 2.7 for this substrate at 0.3 mM Zn2+. The allosteric effect of Zn2+ on the enzyme is not altered by the concentration of carbamoyl phosphate, and the saturation curve of carbamoyl phosphate remains hyperbolic in the presence of the metal ion. At fixed substrate concentrations, initial velocity data obtained at 0.-0.3 mM Zn2+ indicate cooperative binding of the metal ion to ornithine transcarbamoylase; a Hill coefficient of 1.7 +/- 0.1 is found that is independent of the level of L-ornithine. These results suggest competitive and exclusive binding to the enzyme between L-ornithine and Zn2+ with conformational changes induced in the subunits of the enzyme only by the metal ligand. Neither Co2+ nor Cu2+ exerts an effect on the kinetic behavior of the enzyme. This finding reveals not only specific allosteric control of ornithine transcarbamoylase by Zn2+ but also the possibility of an interlocking metabolic regulation between the urea cycle and the pathway for pyrimidine biosynthesis.