Oxygen binding and subunit interaction of hemoglobin in relation to the two-state model

Mills and Ackers (Mills, F.C., and Ackers, G.K. (1979) J. Biol. Chem. 254, 2881-2887) have reported the subunit interactions of hemoglobin to decrease on binding of the fourth molecule of oxygen to hemoglobin. This effect, which they called quaternary enhancement, is incompatible with the two-state Monod, Wyman, and Changeux allosteric model. Their free energy of binding of the fourth molecule (-9.3 kcal/mol) has been compared with independent kinetic estimates which give -8.6 kcal/mol. This smaller value is consistent with literature values and allows reasonable representation of the equilibrium curve using the two-state model without invoking quaternary enhancement.     

Studies on carbon monoxide binding by shark haemoglobin.

The kinetics of the reactions of Pacific-porbeagle haemoglobin with CO were studied by flash-photolysis and stopped-flow methods, and the equilibrium binding curves for CO were measured in spectrophotometric titrations. Measurements were made in the pH range 6-8 and in the temperature range 0-40 degrees C. The results are discussed in terms of the allosteric model proposed by Monod, Wyman & Changeux [(1965) J. Mol. Biol. 12, 88-118]. Within this framework the results indicate that in the R-state the haem groups fall into two classes of different reactivity with different spectral characteristics, but that in the T-state the groups may be essentially equivalent. The physiological importance of the temperature-insensitivity of the equilibrium ligand-binding curves for porbeagle haemoglobin is discussed.     

On the pH-dependence of the reaction of hemoglobin with carbon monoxide.

Flash-photolysis experiments were performed on solutions of carbonmonoxy hemoglobin (human Hb A) as function as pH. The fraction of fast reaction and the amount of photodissociation as produced by a given amount of light quanta has been analyzed in terms of the allosteric model of ligand binding by Monod, Wyman and Changeux. It is shown how the switch-over point of the allosteric transition from the T or R state is controlled by the protons, which act as allosteric effectors.     

Coexpression of alpha and beta subunits of the rod cyclic GMP-gated channel restores native sensitivity to cyclic AMP: role of D604/N1201

Coexpression of the betawt and alphawt subunits of the bovine rod channel restores two characteristics of the native channels: higher sensitivity to cAMP and potentiation of cGMP-induced currents by low cAMP concentrations. To test whether the increased sensitivity to cAMP is due to the uncharged nature of the asparagine residue (N1201) situated in place of aspartate D604 in the beta subunit as previously suggested (, Neuron. 15:619-625), we compared currents from wild-type (alphawt and alphawt/betawt) and from mutated channels (alphaD604N, alphaD604N/betawt, and alphawt/betaN1201D). The results show that the sensitivity to cAMP and cAMP potentiation is partly but not entirely determined by the charge of residue 1201 in the beta subunit. The D604N mutation in the alpha subunit and, to a lesser extent, coexpression of the betawt subunit with the alphawt subunit reduce the open probability for cGMP compared to that of the alphawt channel. Interpretation of the data with the MWC allosteric model (model of Monod, Wyman, Changeux;, J. Mol. Biol. 12:88-118) suggests that the D604N mutation in the alpha subunits and coassembly of alpha and beta subunits alter the free energy of gating by cAMP more than that of cAMP binding.     

ATP inhibition of Phycomyces pyruvate kinase: a kinetic study of the inhibitory effects on the allosteric kinetics shown by the enzyme

Studies on ATP effects on the allosteric kinetics shown by pyruvate kinase from Phycomyces blakesleeanus NRRL 1555 (-) are reported. Phosphoenolpyruvate showed an allosteric ATP-dependent substrate inhibition. The results supported the existence of spatially distinct catalytic binding sites and the inhibitory binding sites for phosphoenolpyruvate, and ATP showed opposite heterotropic effects with respect to these two types of binding site. With respect to Mg2+ ions, ATP caused a negative heterotropic effect. The global inhibitory effect of ATP was in agreement with the predictions postulated by the two-state concerted-symmetry model of Monod, Wyman and Changeux.     

Regulatory properties of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa. Kinetic studies and fluorescence titration.

Mechanisms involved in the action of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa (EC 1.6.1.1) have been investigated by means of kinetic studies and fluorescence titration. Our results, as well as those from previous investigations, suggest that the allosteric MWC model (Monod, J., Wyman, J., and Changeux, J. P. (1965), J. Mol. Biol. 12, 88-118) may be used as a first step for the explanation of the properties of the transhydrogenase. The basic reaction of the enzyme is the oxidation of reduced triphosphopyridine nucleotide (TPNH) by diphosphopyridine nucleotide (DPN+). In terms of the model, the functional R state is favored by TPNH, whereas the product triphosphopyridine nucleotide (TPN+) behaves as an allosteric inhibitor, and is therefore assumed to favor the nonfunctional T state. To a slight extent, the T state is also favored by inorganic phosphate. On the other hand, adenosine 2'-monophosphate and several other 2'-phosphate nucleotides function as activators, and hence are presumed to shift the allosteric equilibrium toward the R state. The studies in this paper suggest a specific regulatory site for the transhydrogenase.     

The model of Monod, Wyman, and Changeux generates two sets of parameters when applied to oxygen binding in hemoglobin

The model of Monod, Wyman and Changeux is applied to binding phenomena where the Mass Law and its expansion are employed. In this communication the model of Monod, Wyman and Changeux (MWC) is applied to analyze the oxygen binding reaction in hemoglobin. The symmetrical structure of the MWC model with its three parameters is such that two sets of these parameters, rather than one, fit experimental data for the binding of oxygen to hemoglobin.     

Pre-steady state quantification of the allosteric influence of Escherichia coli phosphofructokinase.

Stopped-flow kinetics was utilized to determine how allosteric activators and inhibitors of wild-type Escherichia coli phosphofructokinase influenced the kinetic rate and equilibrium constants of the binding of substrate fructose 6-phosphate. Monitoring pre-steady state fluorescence intensity emission changes upon an addition of a ligand to the enzyme was possible by a unique tryptophan per subunit of the enzyme. Binding of fructose 6-phosphate to the enzyme displayed a two-step process, with a fast complex formation step followed by a relatively slower isomerization step. Systematic addition of fructose 6-phosphate to phosphofructokinase in the absence and presence of several fixed concentrations of phosphoenolpyruvate indicated that the inhibitor binds to the enzyme concurrently with the substrate, forming a ternary complex and inducing a conformational change, rather than a displacement of the equilibrium as predicted by the classical two-state model (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118). The activator, MgADP, also altered the affinity of fructose 6-phosphate to the enzyme by forming a ternary complex. Furthermore, both phosphoenolpyruvate and MgADP act by influencing the fast complex formation step while leaving the slower enzyme isomerization step essentially unchanged.     

Thermodynamic studies on oxygen binding by human red blood cells

Oxygen equilibrium curves have been measured on human normal red blood cells, at the temperatures of 20, 25, 30, 37 and 41 degrees C, and at pHs ranging from 6.8 to 8.2. The thermodynamical parameters have been determined for the four successive steps of oxygenation and for overall oxygenation, according to the Adair and MWC models [Monod J, Wyman J, Changeux JP. On the nature of allosteric transitions: a plausible model. J Mol Biol 1965;12:88-118]. The heat release appears to be nearly equal for the four steps. At the first three steps, the delta H change is counterbalanced by a nearly equivalent change of delta S, resulting in a rather small delta G value. delta G is greater at the fourth step, because of diminution of this enthalpy-entropy compensation phenomenon. The four steps are both enthalpy and entropy driven. According to the MWC model, the T to R transition is endothermic, and allosteric quaternary transition occurs at binding of the third oxygen. The average heat release increases by 2.8 kcal/mol when pH raises from 7.4 to 8.2, but flattens below pH 7.4. After correction for the heat of solution of oxygen and for the heat of proton release (referred to intracellular pH), an intrinsic heat for oxygenation of the heme of approximately--13 kcal/mol is obtained for the successive steps of oxygenation (at pH 7.4, 37 degrees C). These results are compared with those previously obtained for pigeon and trout red blood cells.     

Acetylcholine and epibatidine binding to muscle acetylcholine receptors distinguish between concerted and uncoupled models.

The muscle acetylcholine receptor (AChR) has served as a prototype for understanding allosteric mechanisms of neurotransmitter-gated ion channels. The phenomenon of cooperative agonist binding is described by the model of Monod et al. (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118; MWC model), which requires concerted switching of the two binding sites between low and high affinity states. The present study examines binding of acetylcholine (ACh) and epibatidine, agonists with opposite selectivity for the two binding sites of mouse muscle AChRs. We expressed either fetal or adult AChRs in 293 HEK cells and measured agonist binding by competition against the initial rate of 125I-alpha-bungarotoxin binding. We fit predictions of the MWC model to epibatidine and ACh binding data simultaneously, taking as constants previously determined parameters for agonist binding and channel gating steps, and varying the agonist-independent parameters. We find that the MWC model describes the apparent dissociation constants for both agonists but predicts Hill coefficients that are far too steep. An Uncoupled model, which relaxes the requirement of concerted state transitions, accurately describes binding of both ACh and epibatidine and provides parameters for agonist-independent steps consistent with known aspects of AChR function.     

A kinetic study of the pH effect on the allosteric properties of pyruvate kinase from Phycomyces blakesleeanus

This paper reports the pH-dependence of the allosteric kinetics of Phycomyces blakeseeanus pyruvate kinase with phosphoenol pyruvate and Mg2+ ions in the presence and in the absence of fructose 1,6-bisphosphate (allosteric activator) and L-alanine (allosteric inhibitor). Hydrogen ions increase the affinity of the inhibitory binding sites for phosphoenol pyruvate and Mg2+ ions. Assuming partial conformational states of high and low affinity for inhibitory binding sites, the data presented are in good agreement with the predictions postulated by the two-state concerted-symmetry model of Monod, Wyman, and Changeux. Fructose-1,6-bisphosphate and L-alanine show opposite effects on the interactions of phosphoenol pyruvate and Mg2+ ions with their respective catalytic and inhibitory binding sites. At pH 6.0, the regulation of the Phycomyces pyruvate kinase activity by the concentrations of phosphoenol pyruvate and Mg2+ ions is controlled mainly by L-alanine.     

MWC allosteric model explains unusual hemoglobin-oxygen binding curves from sickle cell drug binding

An oxygen-affinity-modifying drug, voxelotor, has very recently been approved by the FDA for treatment of sickle cell disease. The proposed mechanism of action is by preferential binding of the drug to the R quaternary conformation, which cannot copolymerize with the T conformation to form sickle fibers. Here, we report widely different oxygen dissociation and oxygen association curves for normal blood in the presence of voxelotor and interpret the results in terms of the allosteric model of Monod, Wyman, and Changeux with the addition of drug binding. The model does remarkably well in quantitatively explaining a complex data set with just the addition of drug binding and dissociation rates for the R and T conformations. Whereas slow dissociation of the drug from R results in time-independent dissociation curves, the changing association curves result from slow dissociation of the drug from T, as well as extremely slow binding of the drug to T. By calculating true equilibrium curves from the model parameters, we show that there would be a smaller decrease in oxygen delivery from the left shift in the dissociation curve caused by drug binding if drug binding and dissociation for both R and T were rapid. Our application of the Monod, Wyman, and Changeux model demonstrates once more its enormous power in explaining many different kinds of experimental results for hemoglobin. It should also be helpful in analyzing oxygen binding and in vivo delivery in future investigations of oxygen-affinity-modifying drugs for sickle cell disease.     

Integrated allosteric model of voltage gating of HCN channels

Hyperpolarization-activated (pacemaker) channels are dually gated by negative voltage and intracellular cAMP. Kinetics of native cardiac f-channels are not compatible with HH gating, and require closed/open multistate models. We verified that members of the HCN channel family (mHCN1, hHCN2, hHCN4) also have properties not complying with HH gating, such as sigmoidal activation and deactivation, activation deviating from fixed power of an exponential, removal of activation "delay" by preconditioning hyperpolarization. Previous work on native channels has indicated that the shifting action of cAMP on the open probability (Po) curve can be accounted for by an allosteric model, whereby cAMP binds more favorably to open than closed channels. We therefore asked whether not only cAMP-dependent, but also voltage-dependent gating of hyperpolarization-activated channels could be explained by an allosteric model. We hypothesized that HCN channels are tetramers and that each subunit comprises a voltage sensor moving between "reluctant" and "willing" states, whereas voltage sensors are independently gated by voltage, channel closed/open transitions occur allosterically. These hypotheses led to a multistate scheme comprising five open and five closed channel states. We estimated model rate constants by fitting first activation delay curves and single exponential time constant curves, and then individual activation/deactivation traces. By simply using different sets of rate constants, the model accounts for qualitative and quantitative aspects of voltage gating of all three HCN isoforms investigated, and allows an interpretation of the different kinetic properties of different isoforms. For example, faster kinetics of HCN1 relative to HCN2/HCN4 are attributable to higher HCN1 voltage sensors' rates and looser voltage-independent interactions between subunits in closed/open transitions. It also accounts for experimental evidence that reduction of sensors' positive charge leads to negative voltage shifts of Po curve, with little change of curve slope. HCN voltage gating thus involves two processes: voltage sensor gating and allosteric opening/closing.     

Thermodynamic linkages in rabbit muscle pyruvate kinase: kinetic, equilibrium, and structural studies

The mechanism of allosteric regulation of rabbit muscle pyruvate kinase (PK) was examined in the presence of the allosteric inhibitor phenylalanine (Phe). Steady-state kinetic, equilibrium binding, and structural studies were conducted to provide a broad data base to establish a reasonable model for the interactions. Phe was shown to induce apparent cooperativity in the steady-state kinetic measurements at pH 7.5 and 23 degrees C. The apparent Km for phosphoenolpyruvate was shown to increase with increasing Phe concentrations. These results imply that Phe reduces the affinity of PK for phosphoenolpyruvate. This conclusion was substantiated by equilibrium binding studies which yielded association constants of phosphoenolpyruvate as a function of Phe concentration. The binding constant of Phe was also determined at pH 7.0 and 23 degrees C. The effect of ligands on the hydrodynamic properties of PK was monitored by difference sedimentation velocity, sedimentation velocity, and equilibrium experiments. The results showed that PK remains tetrameric both in the presence and in the absence of Phe. However, Phe induces a small decrease in the sedimentation coefficient of the enzyme; hence, it suggests a loosening of the protein structure. The accessibility of the sulfhydryl residues of the enzyme also increases in the presence of Phe. Furthermore, the Phe-induced conformational change was approximately 90% complete when only 25% of the binding sites were saturated. This result suggested that the regulatory behavior of PK might satisfactorily be described by the two-state model of Monod-Wyman-Changeux [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118].     

Subunit interactions in the activation of cyclic nucleotide-gated ion channels.

Cyclic nucleotide-gated (CNG) ion channels of retinal photoreceptors and olfactory neurons are multimeric proteins of unknown stoichiometry. To investigate the subunit interactions that occur during CNG channel activation, we have used tandem cDNA constructs of the rod CNG channel to generate heteromultimeric channels composed of wild-type and mutant subunits. We introduced point mutations that affect channel activation: 1) D604M, which alters the relative ability of agonists to promote the allosteric conformational change(s) associated with channel opening, and 2) T560A, which primarily affects the initial binding affinity for cGMP, and to a lesser extent, the allosteric transition. At saturating concentrations of agonist, heteromultimeric channels were intermediate between wild-type and mutant homomultimers in agonist efficacy and apparent affinity for cGMP, cIMP, and cAMP, consistent with a model for the allosteric transition involving a concerted conformational change in all of the channel subunits. Results were also consistent with a model involving independent transitions in two or three, but not one or four, of the channel subunits. The behavior of the heterodimers implies that the channel stoichiometry is some multiple of 2 and is consistent with a tetrameric quaternary structure for the functional channel complex. Steady-state dose-response relations for homomultimeric and heteromultimeric channels were well fit by a Monod, Wyman, and Changeux model with a concerted allosteric opening transition stabilized by binding of agonist.     

Allosteric voltage gating of potassium channels II. Mslo channel gating charge movement in the absence of Ca(2+).

Large-conductance Ca(2+)-activated K(+) channels can be activated by membrane voltage in the absence of Ca(2+) binding, indicating that these channels contain an intrinsic voltage sensor. The properties of this voltage sensor and its relationship to channel activation were examined by studying gating charge movement from mSlo Ca(2+)-activated K(+) channels in the virtual absence of Ca(2+) (<1 nM). Charge movement was measured in response to voltage steps or sinusoidal voltage commands. The charge-voltage relationship (Q-V) is shallower and shifted to more negative voltages than the voltage-dependent open probability (G-V). Both ON and OFF gating currents evoked by brief (0.5-ms) voltage pulses appear to decay rapidly (tau(ON) = 60 microseconds at +200 mV, tau(OFF) = 16 microseconds at -80 mV). However, Q(OFF) increases slowly with pulse duration, indicating that a large fraction of ON charge develops with a time course comparable to that of I(K) activation. The slow onset of this gating charge prevents its detection as a component of I(gON), although it represents approximately 40% of the total charge moved at +140 mV. The decay of I(gOFF) is slowed after depolarizations that open mSlo channels. Yet, the majority of open channel charge relaxation is too rapid to be limited by channel closing. These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C-C, O-O, and C-O transitions.     

Comments on Extensions of the Allosteric Model for Haemoglobin

RECENTLY Edelstein¹ has concluded on the basis of a numerical analysis that the sequential model as formulated by Koshland, Nemethy and Filmer (KNF)² describes the oxygen binding curves of a number of species of human haemoglobin less well than does the two-state allosteric model of Monod, Wyman and Changeux (MWC)³. This communication demonstrates that Edelstein's analysis is incomplete and that extension of his analysis reveals that no such conclusion can be drawn from the data considered.     

Kinetics of ligand binding and quaternary conformational change in the homodimeric hemoglobin from Scapharca inaequivalvis

The kinetics of the reaction with oxygen and carbon monoxide of the homodimeric hemoglobin from the bivalve mollusc Scapharca inaequivalvis has been extensively investigated by flash and dye-laser photolysis, temperature jump relaxation, and stopped flow methods. The results indicate that cooperativity in ligand binding, already observed for oxygen at equilibrium, finds its kinetic counterpart in a large decrease of the oxygen dissociation velocity in the second step of the binding reaction. In the case of carbon monoxide, cooperativity is clearly evident in the increase of the combination velocity constant as the reaction proceeds. Therefore, the ligand-binding kinetics of this dimeric hemoglobin shows the characteristic features of the corresponding reactions of tetrameric hemoglobins. Analysis of the data in terms of the allosteric model proposed by Monod et al. (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118) has shown that the values of the allosteric parameters cannot be fixed uniquely for a dimeric hemoglobin. The rapid changes in absorbance observed at the isosbestic points of unliganded and liganded hemoglobin following laser photolysis provided a value of 7 X 10(4) S-1 at 20 degrees C for the rate of the ligand-free quarternary conformational change, postulated on the basis of cooperative ligand binding. Comparison of the rapid absorbance changes observed during ligand rebinding in this hemoglobin with those observed in tuna hemoglobin indicate that, at full photolysis, binding to the T state is followed by further binding and conversion to the liganded R state; at partial photolysis, population of the liganded T state occurs immediately and is followed by a decay to the liganded R state upon further ligand binding. These new results, in conjunction with previous equilibrium data on the same system, show unequivocally that the presence of two different types of chain is not an absolute prerequisite for cooperativity in hemoglobins, contrary to currently accepted ideas.