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PubMed1978

[Allosteric regulation of ceruloplasmin activity].

Study of the interactions of homogenous human ceruloplasmin preparations with histamine show that the rate of p-phenylene diamine oxidation by ceruloplasmin is increased in the presence of histamine; the increase in the enzyme activity is independent of histamine concentration. The dependence of the reaction rate on substrate concentration is S-shaped, both in the presence and in the absence of histamine. The respective values of the Hill coefficient and Rs for the enzyme in the presence and in the absence of histamine are 2.5 and 2.0 and 8.0 and 10.4. Histamine does not change ceruloplasmin-specific absorption at 610 nm. Evidence from EPR studies show that histamine does not interact with Cu of the enzyme active center. During interaction with histamine the antigenic properties of the enzyme are changed. Histamine increases the oxidase activity of the enzyme in human and rat blood sera and exerts multifold effects on the enzyme activity in patients with hepatolenticular degeneration. After injection of histamine to rats the enzyme activity is increased without a simultaneous increase in Cu concentration in the blood serum, i.e. without de novo synthesis of ceruloplasmin. The data obtained suggest that ceruloplasmin is probably an allosteric enzyme, which histamine is its positive allosteric effector.

Allosteric Regulation↗
PubMed2006

Ligand-specific allosteric regulation of coactivator functions of androgen receptor in prostate cancer cells.

The androgen receptor not only mediates prostate development but also serves as a key regulator of primary prostatic cancer growth. Although initially responsive to selective androgen receptor modulators (SARMs), which cause recruitment of the nuclear receptor-corepressor (N-CoR) complex, resistance invariably occurs, perhaps in response to inflammatory signals. Here we report that dismissal of nuclear receptor-corepressor complexes by specific signals or androgen receptor overexpression results in recruitment of many of the cohorts of coactivator complexes that permits SARMs and natural ligands to function as agonists. SARM-bound androgen receptors appear to exhibit failure to recruit specific components of the coactivators generally bound by liganded nuclear receptors, including cAMP response element-binding protein (CBP)/p300 or coactivator-associated arginine methyltransferase 1 (CARM1) to the SARM-bound androgen receptor, although still causing transcriptional activation of androgen receptor target genes. SARM-bound androgen receptors use distinct LXXLL (L, leucine; X, any amino acid) helices in the p160 nuclear receptor interaction domains that may impose selective allosteric effects, providing a component of the molecular basis of differential responses to different classes of ligands by androgen receptor.

Allosteric Regulation↗
PubMed2003

Allosteric regulation of the transcription factor NFAT1 by multiple phosphorylation sites: a mathematical analysis.

NFAT transcription factors are activated through dephosphorylation by the phosphatase calcineurin. Experimental data show that 13 conserved phosphorylation sites conspire to control the transition between an inactive and an active conformation. We propose a quantitative model of the underlying molecular mechanisms that may generally apply to highly phosphorylated proteins. Mathematical analysis shows that multiple phosphorylation sites result in a threshold for protein activation. Its sharpness increases with the number of sites, thus providing a rationale for the involvement of the large number of serine residues in NFAT activation. The model predicts that nuclear kinases exert a larger control on the activation threshold than cytoplasmic kinases, and that the NFAT activation kinetics can discriminate between input signals of different amplitude.

Allosteric Regulation↗
PubMed1997

A structural determinant of desensitization and allosteric regulation by pentobarbitone of the GABAA receptor.

Functional properties of the alpha1beta1 GABAA receptor changes in a subunit-specific manner when a threonine residue in the M2 region at the 12' position was mutated to glutamine. The rate and extent of desensitization increased in all mutants but the rate of activation was faster in the beta1 mutants. A negligible plateau current and abolition of potentiation by pentobarbitone of the GABA-activated current depended on the Thr 12' Gln mutation being present in the beta1 subunit. The Hill coefficient of the peak current response to GABA was reduced to less than one also in a beta1 subunit-specific manner. It was concluded that the beta1 subunit dominated conformational changes activated by GABA.

Allosteric Regulation↗
PubMed2002

The archaeogastropod mollusc Haliotis iris: tissue and blood metabolites and allosteric regulation of haemocyanin function.

We investigated divalent cation and anaerobic end-product concentrations and the interactive effects of these substances and pH on haemocyanin oxygen-binding (Hc-O(2)) in the New Zealand abalone Haliotis iris. During 24 h of environmental hypoxia (emersion), D-lactate and tauropine accumulated in the foot and shell adductor muscles and in the haemolymph of the aorta, the pedal sinus and adductor muscle lacunae, whereas L-lactate was not detected. Intramuscular and haemolymph D-lactate concentrations were similar, but tauropine accumulated to much higher levels in muscle tissues. Repeated disturbance and short-term exposure to air over 3 h induced no accumulation of D- or L-lactate and no change in [Ca(2+)], [Mg(2+)], pH and O(2)-binding properties of the native haemolymph. The haemolymph showed a low Hc-O(2) affinity, a large reverse Bohr effect and marked cooperativity. Dialysis increased Hc-O(2) affinity, obliterated cooperativity and decreased the pH-sensitivity of O(2) binding. Replacing Mg(2+) and Ca(2+) restored the native O(2)-binding properties and the reverse Bohr shift. L- and D-lactate exerted minor modulatory effects on O(2)-affinity. At in vivo concentrations of Mg(2+) and Ca(2+), the cooperativity is dependent largely on Mg(2+), which modulates the O(2) association equilibrium constants of both the high-affinity (K(R)) and the low-affinity (K(T)) states (increasing and decreasing, respectively). This allosteric mechanism contrasts with that encountered in other haemocyanins and haemoglobins. The functional properties of H. iris haemocyanin suggest that high rates of O(2) delivery to the tissues are not a priority but are consistent with the provision of a large O(2) reserve for facultatively anaerobic tissues during internal hypoxia associated with clamping to the substratum.

Allosteric Regulation↗
PubMed2004

Phosphoprotein inhibitor CPI-17 specificity depends on allosteric regulation of protein phosphatase-1 by regulatory subunits.

Inhibition of myosin phosphatase is critical for agonist-induced contractility of vascular smooth muscle. The protein CPI-17 is a phosphorylation-dependent inhibitor of myosin phosphatase and, in response to agonists, Thr-38 is phosphorylated by protein kinase C, producing a >1,000-fold increase in inhibitory potency. Here, we addressed how CPI-17 could selectively inhibit myosin phosphatase among other protein phosphatase-1 (PP1) holoenzymes. PP1 in cell lysates was separated by sequential affinity chromatography into at least two fractions, one bound specifically to thiophospho-CPI-17, and another bound specifically to inhibitor-2. The MYPT1 regulatory subunit of myosin phosphatase was concentrated only in the fraction bound to thiophospho-CPI-17. This binding was eliminated by addition of excess microcystin-LR to the lysate, showing that binding at the active site of PP1 is required. Phospho-CPI-17 failed to inhibit glycogen-bound PP1 from skeletal muscle, composed primarily of PP1 with the striated muscle glycogen-targeting subunit (G(M)) regulatory subunit. Phospho-CPI-17 was dephosphorylated during assay of glycogen-bound PP1, not MYPT1-associated PP1, even though these two holoenzymes have the same PP1 catalytic subunit. Phosphorylation of CPI-17 in rabbit arteries was enhanced by calyculin A but not okadaic acid or fostriecin, consistent with PP1-mediated dephosphorylation. We propose that CPI-17 binds at the PP1 active site where it is dephosphorylated, but association of MYPT1 with PP1C allosterically retards this hydrolysis, resulting in formation of a complex of MYPT1.PP1C.P-CPI-17, leading to an increase in smooth muscle contraction.

Allosteric Regulation↗
PubMed1989

Allosteric regulation of yeast inorganic pyrophosphatase by substrate.

Kinetic and binding studies of yeast inorganic pyrophosphatase (EC 3.6.1.1) revealed a regulatory PPi-binding site. Rate vs substrate concentration dependencies were markedly nonhyperbolic in the range of 0.1-150 microM MgPPi at fixed Mg2+ levels of 0.05-10 mM provided that the enzyme had been preequilibrated with Mg2+. Imidodiphosphate, hydroxymethylenebisphosphonate, and phosphate eliminated the deviations from the Michaelis-Menten kinetics and inhibited PPi hydrolysis in a manner consistent with their binding at both active and regulatory sites. The results agreed with a model in which binding of uncomplexed PPi at the regulatory site markedly increases enzyme affinity for the activating Mg2+ ion. Ultrafiltration studies revealed the binding of at least 3 mol of the inhibitory hydroxymethylenebisphosphonate and of 2 mol of noninhibitory methylenebisphosphonate per mole of the dimeric enzyme.

Allosteric Regulation↗
PubMed1993

Block of cardiac ATP-sensitive K+ channels by external divalent cations is modulated by intracellular ATP. Evidence for allosteric regulation of the channel protein.

We have investigated the interactions between extracellular divalent cations and the ATP-sensitive potassium channel in single guinea pig ventricular cells and found that, under whole-cell patch clamp recording conditions, extracellularly applied Co2+, Cd2+, and Zn2+ block current through the ATP-sensitive K channel (IKATP). The respective Kd's for block of IKATP by Cd2+ and Zn2+ are 28 and 0.46 microM. The Kd for Co2+ is > 200 microM. Extracellular Ca2+ and Mg2+ appear to have no effect at concentrations up to 1 and 2 mM, respectively. Block of IKATP by extracellular cations is not voltage dependent, and both onset and recovery from block occur within seconds. Single-channel experiments using the inside-out patch configuration show that internally applied Cd2+ and Zn2+ are not effective blockers of IKATP. Experiments in the outside-out patch configuration confirm that the divalent cations interact directly with IKATP channel activity. Our study also shows that this block of IKATP is dependent on intracellular ATP concentrations. Under whole-cell conditions, when cells are dialyzed with [ATP]pipette = 0, the degree of cation block is reduced. This dependence on intracellular ATP was confirmed at the single-channel level by experiments in excised, inside-out patch configurations. Our results show that some, but not all, divalent cations inhibit current through IKATP channels by binding to sites that are not within the transmembrane electric field, but are on the extracellular membrane surface. The interdependence of internal ATP and external divalent cation binding is consistent with an allosteric interaction between two binding sites and is highly suggestive of a modulatory mechanism involving conformational change of the channel protein.

Adenosine Triphosphate↗
PubMed2002

Conformational switch between slow and fast gating modes: allosteric regulation of voltage sensor mobility in the EAG K+ channel.

Voltage-gated EAG K+ channels switch between fast and slow gating modes in a Mg2+-dependent manner by an unknown mechanism. We analyzed molecular motions in and around the voltage-sensing S4 in bEAG1. Using accessibility and perturbation analyses, we found that activation increases both the charge occupancy and volume of S4 side chains in the gating canal. Fluorescence measurements suggest that mode switching is due to a motion of the S2/S3 side of the gating canal. We propose that when S4 is in the resting state and its thin end is in the gating canal, a conformational rearrangement of S2/S3 narrows the canal around S4, forming the Mg2+ binding site. Binding of Mg2+ is proposed to stabilize this conformation and to slow opening of the gate by impeding S4's voltage-sensing outward motion.

Allosteric Regulation↗
PubMed2001

Allosteric regulation of substrate binding and product release in geranylgeranyltransferase type II.

GTPases of the Rab family are key components of vesicular transport in eukaryotic cells. Posttranslational attachment of geranylgeranyl moieties is essential for Rab function. Geranylgeranyltransferase type II (GGTase-II) catalyzes the modification of Rab proteins once they are in complex with their escort protein (REP). Upon completion of prenylation, REP and modified Rab leave the enzyme, enabling a new round of catalysis. We have studied the mechanism underlying substrate binding and product release in the geranylgeranylation of Rab proteins. Binding of the Rab7:REP-1 complex to GGTase-II was found to be strongly modulated by geranylgeranyl pyrophosphate (GGpp). The affinity of GGTase-II for the Rab7:REP-1 complex increases from ca. 120 nM to ca. 2 nM in the presence of GGpp. To study the effect of GGpp on interaction of the enzyme with its product, we generated semisynthetic doubly prenylated Rab7 bearing a fluorescent reporter group. Using this novel compound, we demonstrated that the affinity of doubly prenylated Rab7:REP-1 complex for GGTase-II was 2 and 18 nM in the absence and presence of GGpp, respectively. The difference in affinities originates mainly from a difference in the dissociation rates. Thus, binding of the new isoprenoid substrate molecule facilitates the product release by GGTase-II. The affinity of GGpp for the prenylated Rab7:REP-1:GGTase-II was K(d) = 22 nM, with one molecule of GGpp binding per molecule of prenylated ternary complex. We interpreted this finding as an indication that the geranylgeranyl moieties transferred to Rab protein do not occupy the GGpp binding site of the GGTase-II. In summary, these results demonstrate that GGpp acts as an allosteric activator that stabilizes the Rab7:REP-1:GGTase-II complex and triggers product release upon prenylation, preventing product inhibition of the enzyme.

Adaptor Proteins, Signal Transducing↗
PubMed1987

[Allosteric regulation of Ca2+-calmodulin-dependent phosphodiesterase activity from the bovine brain].

The Ca2+-calmodulin-dependent interaction of phosphodiesterase with phenyl-Sepharose was demonstrated. BSA caused incomplete competitive inhibition of phosphodiesterase activation by calmodulin. The 17-fold increase of the constant for phosphodiesterase activation by calmodulin was accompanied by an insignificant rise in the maximum rate of cAMP hydrolysis; in this case the value of the inhibition constant amounted to Ki approximately 6 microM. In the absence of calmodulin saturating concentrations of BSA reduced the enzyme activity nearly 3-4-fold. The effect of BSA on phosphodiesterase was incompetitive with respect to cAMP (Ki approximately 1.4 microM). Both phenomena are characteristic of incompetitive binding of BSA to the enzyme with respect to cAMP and calmodulin. Gel filtration data reflect the changes in the enzyme molecular weight during its interaction with BSA. All the above reactions of the enzyme are reversible.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2001

The gdhB gene of Pseudomonas aeruginosa encodes an arginine-inducible NAD(+)-dependent glutamate dehydrogenase which is subject to allosteric regulation.

The NAD(+)-dependent glutamate dehydrogenase (NAD-GDH) from Pseudomonas aeruginosa PAO1 was purified, and its amino-terminal amino acid sequence was determined. This sequence information was used in identifying and cloning the encoding gdhB gene and its flanking regions. The molecular mass predicted from the derived sequence for the encoded NAD-GDH was 182.6 kDa, in close agreement with that determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme (180 kDa). Cross-linking studies established that the native NAD-GDH is a tetramer of equal subunits. Comparison of the derived amino acid sequence of NAD-GDH from P. aeruginosa with the GenBank database showed the highest homology with hypothetical polypeptides from Pseudomonas putida, Mycobacterium tuberculosis, Rickettsia prowazakii, Legionella pneumophila, Vibrio cholerae, Shewanella putrefaciens, Sinorhizobium meliloti, and Caulobacter crescentus. A moderate degree of homology, primarily in the central domain, was observed with the smaller tetrameric NAD-GDH (protomeric mass of 110 kDa) from Saccharomyces cerevisiae or Neurospora crassa. Comparison with the yet smaller hexameric GDH (protomeric mass of 48 to 55 kDa) of other prokaryotes yielded a low degree of homology that was limited to residues important for binding of substrates and for catalytic function. NAD-GDH was induced 27-fold by exogenous arginine and only 3-fold by exogenous glutamate. Primer extension experiments established that transcription of gdhB is initiated from an arginine-inducible promoter and that this induction is dependent on the arginine regulatory protein, ArgR, a member of the AraC/XyIS family of regulatory proteins. NAD-GDH was purified to homogeneity from a recombinant strain of P. aeruginosa and characterized. The glutamate saturation curve was sigmoid, indicating positive cooperativity in the binding of glutamate. NAD-GDH activity was subject to allosteric control by arginine and citrate, which function as positive and negative effectors, respectively. Both effectors act by influencing the affinity of the enzyme for glutamate. NAD-GDH from this organism differs from previously characterized enzymes with respect to structure, protomer mass, and allosteric properties indicate that this enzyme represents a novel class of microbial glutamate dehydrogenases.

Allosteric Regulation↗
PubMed1993

Precoupling of Gi/G(o)-linked receptors and its allosteric regulation by monovalent cations.

The ability of receptors (R) to activate G-proteins (G) is promoted by the binding of agonists, reflecting their induction of a receptor conformation which facilitates both the formation of a RG complex and guanine nucleotide exchange. Recent evidence from isolated membrane studies has indicated, however, that some receptors have the inherent ability to form RG complexes and promote GDP/GTP exchange in their unoccupied state. These receptors preferentially activate pertussis toxin-sensitive G-proteins (i.e. Gi/G(o)) and the interactions of R and G are modulated by monovalent cations (most notably Na+) both in the unoccupied and agonist-occupied states. Basal G-protein activation by such receptors is reduced both by increasing levels of cation and by antagonists which may act by inducing receptor conformations which are less favorable for RG complexation. This behaviour conforms to the predictions of a ternary complex model of receptor function and can be considered in structural terms for those receptors such as the alpha-2 adrenergic receptor where ligand binding and G-protein activation regions have been proposed.

Allosteric Regulation↗
PubMed1999

Allosteric regulation of glycogen synthase and hexokinase by glucosamine-6-phosphate during glucosamine-induced insulin resistance in skeletal muscle and heart.

Glucosamine infusion induces insulin resistance in vivo, but the effect of glucosamine on intracellular metabolites of the hexosamine pathway, especially glucosamine-6-phosphate (GlcN6P) is unknown. Because of the structural similarity of glucose-6-phosphate (G-6-P) and GlcN6P, we hypothesized that accumulation of this metabolite might alter the activities of enzymes such as glycogen synthase and hexokinase. We infused glucosamine (30 micromol x kg(-1) x min(-1)) to induce insulin resistance in rats during a euglycemic-hyperinsulinemic clamp. Glucosamine induced whole-body insulin resistance, which was apparent after 90 min and continued progressively for 360 min. Despite inducing severe whole-body insulin resistance and decrease in glycogen synthase fractional activity in rectus abdominis muscle (69+/-3 vs. 83+/-1%, P<0.01) and heart (7+/-1 vs. 32+/-4%, P<0.001), glucosamine did not change the glycogen content in rectus and even increased it in the heart (209+/-13 vs. 117+/-9 mmol/kg dry wt, P<0.001). Glucosamine increased tissue concentrations of UDP-GlcNAc 4.4- and 4.6-fold in rectus abdominis and heart, respectively. However, GlcN6P concentrations increased 500- and 700-fold in glucosamine-infused animals in rectus abdominis (590+/-80 vs. 1.2+/-0.1 micromol/kg wet wt, P<0.001) and heart (7,703+/-993 vs. 11.2+/-2.3 micromol/kg wet wt, P<0.001). To assess the possible significance of GlcN6P accumulation, we measured the effect of GlcN6P on glycogen synthase and hexokinase activity in vitro. At the GlcN6P concentrations measured in rectus abdominis and heart in vivo, glycogen synthase was activated by 21 and 542%, while similar concentrations inhibited hexokinase activity by 5 and 46%, respectively. This study demonstrates that infusion of glucosamine during a euglycemic-hyperinsulinemic clamp results in marked accumulation of intracellular GlcN6P. The GlcN6P concentrations in the heart and rectus abdominis muscle reach levels sufficient to cause allosteric activation of glycogen synthase and inhibition of hexokinase.

Allosteric Regulation↗
PubMed2001

Allosteric regulation provides a molecular mechanism for preferential utilization of the fully assembled dolichol-linked oligosaccharide by the yeast oligosaccharyltransferase.

The oligosaccharyltransferase (OST) preferentially utilizes the fully assembled dolichol-linked oligosaccharide Glc(3)Man(9)GlcNAc(2)-PP-Dol as the donor for N-linked glycosylation of asparagine residues in N-X-T/S consensus sites in newly synthesized proteins. A wide variety of assembly intermediates (Glc(0-2)Man(0-9)GlcNAc(2)-PP-Dol) can serve as the donor substrate for N-linked glycosylation of peptide acceptor substrates in vitro or of nascent glycoproteins in mutant cells that are defective in donor substrate assembly. A kinetic mechanism that can account for the selection of the fully assembled donor substrate from a complex mixture of dolichol-linked oligosaccharides (OS-PP-Dol) has not been elucidated. Here, the steady-state kinetic properties of the OST were reinvestigated using a proteoliposome assay system consisting of the purified yeast enzyme, near-homogeneous preparations of a dolichol-linked oligosaccharide (Glc(3)Man(9)GlcNAc(2)-PP-Dol or Man(9)GlcNAc(2)-PP-Dol) and an (125)I-labeled tripeptide as the acceptor substrate. The K(m) of the OST for the acceptor tripeptide was only slightly enhanced when Glc(3)Man(9)GlcNAc(2)-PP-Dol was the donor substrate relative to when Man(9)GlcNAc(2)-PP-Dol was the donor substrate. Evaluation of the kinetic data for both donor substrates showed deviations from typical Michaelis-Menten kinetics. Sigmoidal saturation curves, Lineweaver-Burk plots with upward curvature, and apparent Hill coefficients of about 1.4 suggested a substrate activation mechanism involving distinct regulatory (activator) and catalytic binding sites for OS-PP-Dol. Results of competition experiments using either oligosaccharide donor as an alternative substrate were also consistent with this hypothesis. We propose that binding of either donor substrate to the activator site substantially enhances Glc(3)Man(9)GlcNAc(2)-PP-Dol occupancy of the enzyme catalytic site via allosteric activation.

Allosteric Regulation↗
PubMed1978

Subunit dissociation in the allosteric regulation of glycerol kinase from Escherichia coli. 2. Physical evidence.

The dependence of the molecular weight of glycerol kinase on enzyme concentration and on binding of fructose 1,6-bisphosphate has been examined by velocity sedimentation, gel filtration, and polyacrylamide gel electrophoresis. The sedimentation coefficient and Stokes radius decrease as a consequence of dilution in a manner consistent with dissociation into half-molecules, with limiting values suggesting molecular weights of about 218,000 and 136,000 for the associated and dissociated species, respectively. Fructose 1,6-bisphosphate (5 mM) prevents the decrease in sedimentation coefficient brought about by dilution, suggesting a decrease in the apparent subunit dissociation constant of at least four orders of magnitude. Electrophoretic mobility in polyacrylamide gels increases as a consequence of dilution in the absence, but not in the presence, of fructose 1,6-bisphosphate. Ferguson plots indicate that glycerol kinase has the same molecular weight in the presence of fructose 1,6-bisphosphate as the covalently cross-linked tetramer and is substantially smaller in the absence of fructose 1,6-bisphosphate. These results are consistent with the model of glycerol kinase proposed in the preceding paper of this issue [de Riel, J.K., and Paulus, H. (1978), Biochemistry 17] relating subunit dissociation and ligand binding.

Allosteric Regulation↗
PubMed1992

Allosteric regulation of rat testis mitochondrial aldehyde dehydrogenase by capronaldehyde and magnesium ion.

1. The influence of Mg2+ on the kinetic behaviour of mitochondrial aldehyde dehydrogenase from rat testis has been investigated using capronaldehyde as substrate. 2. The kinetic data, obtained by numerical analysis of the progress curves of aldehyde oxidation, were fitted to a modified version of the Monod-Wyman-Changeux model and the fitting procedure resulted in a good correspondence between theoretical and experimental reaction rates over a wide range of capronaldehyde and Mg2+ concentrations. 3. According to the model, the tetrameric enzyme is in equilibrium between two conformational states R and T which display comparable affinities for capronaldehyde (the dissociation constants are 0.17 and 0.3 microM, respectively), but different catalytic power (VT = 2VR). The T state can bind with lower affinity a second molecule of aldehyde (K = 2.5 microM). 4. Mg2+ stabilizes the T state (the dissociation constants for the R and T states are 2.2 and 0.12 mM, respectively) and acts as a strong activator of the R state, but as a weak inhibitor of the T state. In the absence of substrates and Mg2+, the R<-->T equilibrium favors the R state ([T]/[R] = 0.16). 5. The model is able to predict the kinetic behaviour also when the NAD+ concentrations are not saturating and when inhibitory effects by NADH are taken into account.

Aldehyde Dehydrogenase↗
PubMed2000

Confirmation of a unique intra-dimer cooperativity in the human hemoglobin alpha(1)beta(1)half-oxygenated intermediate supports the symmetry rule model of allosteric regulation.

The contribution of the alpha(1)beta(1)half-oxygenated tetramer [alphabeta:alphaO(2)betaO(2)] (species 21) to human hemoglobin cooperativity was evaluated using cryogenic isoelectric focusing. The cooperative free energy of binding, reflecting O(2)-driven protein structure changes, was measured as (21)DeltaG(c) = 5.1 +/- 0. 3 kcal for the Zn/FeO(2) analog. For the Fe/FeCN analog, (21)DeltaG(c) was estimated as 4.0 kcal after correction for a CN ligand rearrangement artifact, demonstrating that ligand rearrangement does not invalidate previous conclusions regarding this species. In the context of the entire Hb cooperativity cascade, which includes eight intermediate species, the 21 tetramer is highly abundant relative to the other doubly-ligated species, providing strong support for the previously determined consensus partition function of O(2) binding and for the Symmetry Rule model of hemoglobin cooperativity (Ackers et al., Science 1992;255:54-63). Cooperativity of normal human hemoglobin is shown to depend on site-configuration, and not solely the number of O(2) bound, nor the occupancy of alpha vs. beta subunits. Verification of a unique contribution from the alpha(1)beta(1)doubly-oxygenated species to the equilibrium O(2) binding curve strongly reinforces the Symmetry Rule interpretation that the alpha(1)beta(1)dimer acts both as a structural and functional element in cooperative O(2) binding.

Allosteric Regulation↗