Cooperative Ligand Binding Research Articles

Evolution of cooperativity in hemoglobins: What can invertebrate hemoglobins tell us?

While vertebrate hemoglobins typically are tetrameric and show highly regulated and cooperative ligand binding, little is known of the evolution of these properties. We are studying the structural and functional properties of the hemoglobins from Caudina arenicola, an echinoderm. The echinoderms are in the lineage most closely related to the vertebrates to express hemoglobin. C. arenicola has three sets of red cells, in the water vascular system, the coelomic cavity, and in an intestinal vein. Each of these expresses a distinct array of globins. The hemoglobins are cooperative and exhibit unusual ligand-linked associative properties, being dimeric when oxygenated and forming tetramers and higher aggregates on deoxygenation. The major coelomic hemoglobins have been subjected to a detailed examination by a combination of ligand binding analyses and protein and DNA sequencing, as well as X-ray crystallography. Two typical globin introns were identified, along with a unique intron that bisects an N-terminal extension of the globin from the remainder of the gene. X-ray crystallographic analysis shows that the subunit interfaces of C. arenicola hemoglobins differ radically from those of vertebrate hemoglobins and indeed from some other invertebrate hemoglobins, but closely resemble the packing arrangements found in a clam hemoglobin (Scapharca). However, the residues implicated in cooperativity in these two types of hemoglobins differ substantially.

Electrostatic coupling to pH-titrating sites as a source of cooperativity in protein-ligand binding.

This paper describes an alternative mechanism for the cooperative binding of charged ligands to proteins. The ligand-binding sites are electrostatically coupled to protein side chains that can undergo protonation and deprotonation. The binding of one ligand alters the protein's protonation equilibrium in a manner that makes the the binding of the second ligand more favorable. This mechanism requires no conformational change to produce a cooperative effect, although it is not exclusive of conformational change. We present a theoretical description of the mechanism, and calculations on three kinds of systems: A model system containing one protonation site and two ligand-binding sites; a model system containing two protonation sites and two ligand-binding sites; and calbindin D9k, which contains two Ca2+-binding sites and 30 protonation sites. For the one-protonation-site model, it is shown that the influence of the protonation site can only be cooperative. The competition of this effect with the anticooperative effect of ligand-ligand repulsion is studied in detail. For the two-protonation site model, the effect can be either cooperative or, in special cases, anticooperative. For calbindin D9k, the calculations predict that six protonation sites in or near the ligand-binding sites make a cooperative contribution that approximately cancels the anticooperative effect of Ca2+-Ca2+ repulsion, accounting for more than half of the total cooperative effect that is needed to overcome repulsion and produce the net cooperativity observed experimentally. We argue that cooperative mechanisms of the kind described here are likely when there is more than one ligand-binding site in a protein domain.

Cooperativity in Bacillus stearothermophilus pyruvate kinase

The enzyme pyruvate kinase (PK) from the moderate thermophile Bacillus stearothermophilus has been used as a model system with which to investigate the homotropic and heterotropic cooperative interactions of the enzyme. Cooperative ligand binding by the wild-type enzyme was measured using pre-steady-state and steady-state fluorescence spectroscopy, and steady-state kinetics. The results suggest that the cooperative structural changes induced by the substrate phosphoenolpyruvate (PEP) are distinct from those induced by the allosteric activator ribose-5-phosphate (R5P). Furthermore the structural transition induced by the binding of saturating amounts of both PEP and R5P is itself distinct. This conclusion was further substantiated by the production of five mutant proteins in which the R5P- and PEP-induced homotropic cooperative transitions were separated. These results suggest that the cooperativity exhibited by pyruvate kinase from B. stearothermophilus does not conform to a simple two-state model. A putative four-state model is proposed.

Cooperative mechanism in the homodimeric myoglobin from Nassa mutabilis.

Oxygen binding and spectroscopic properties of the homodimeric myoglobin (Mb) from the prosobranchia sea snail Nassa mutabilis have been investigated. Oxygen equilibrium curves are pH-independent and cooperative with P50 = 5 +/- 1 mmHg and n approximately 1.5. Circular dichroism spectra of the oxygenated and deoxygenated form of N. mutabilis Mb are superimposable between 190 and 250 nm, suggesting a mechanism for cooperative ligand binding that does not involve changes in the alpha-helical content of the whole protein. The oxygen dissociation process is biphasic and pH-dependent, with different pKa values (=6.7 +/- 0.2 and 8.5 +/- 0.3) for the two phases. Moreover, the activation energy is essentially the same for both oxygen dissociation processes (Ea = 56.4 +/- 2.1 kJ/mol for the fast phase, and Ea = 53.8 +/- 1.9 kJ/mol for the slow phase), indicating that the rate difference for O2 dissociation between the diliganded and the monoliganded species is mostly dependent on a variation of the activation entropy. Ferrous nitrosylated N. mutabilis Mb shows, at alkaline and neutral pH, axial and rhombic X-band EPR signals, respectively, which display below pH 6 a three-hyperfine pattern typical of five-coordination. The results presented here suggest that in N.mutabilis Mb the kinetic control of cooperativity operates through a mechanism never observed before in other hemoproteins, which requires a ligand-linked large enhancement for the value of the oxygen association process in a molecule not undergoing changes in quaternary structure.

Cooperative ligand binding by bovine phenol sulfotransferase

Although several phenol sulfotransferases (PSTs) that metabolize hormones and xenobiotics have been purified and examined by steady state kinetic methods, little is known about ligand binding and subunit interactions in these enzymes. Inhibition of a purified recombinant homodimeric bovine PST by 2,6-dichloro-4-nitrophenol (DCNP) and pentachlorophenol (PCP) displayed very sharp titration curves that required modeling with Hill equations with slope factors of 2 and 3, respectively. This observation suggested positive cooperative ligand binding during catalytic turnover. The binding of PCP was also monitored by intrinsic protein fluorescence, which was quenched up to 36% upon saturation with the inhibitor. In the absence of 3′-phosphoadenosine-5′-phosphosulfate (PAPS), quenching curves were fit with the Hill equation and an interaction factor of 1. In contrast, inclusion of PAPS increased the association of PCP and resulted in positive cooperative binding with an interaction factor of 1.6–1.9. Whereas adenosine-3′,5′-diphosphate (PAP) also facilitated cooperative binding of PCP, adenosine-5′-monophosphate (AMP) was not effective. This correlated to inhibition of PST by PAP, whereas AMP was not inhibitory up to 1 mM. Therefore, occupation of the PST nucleotide binding site(s) facilitates a subunit interaction that can promote subsequent binding of phenolic ligands.

Interactive and Dominant Effects of Residues 128 and 141 on Cyclic Nucleotide and DNA Bindings in Escherichia coli cAMP Receptor Protein

The molecular events in the cAMP-induced allosteric activation of cAMP receptor protein (CRP) involve interfacial communications between subunits and domains. However, the roles of intersubunit and interdomain interactions in defining the selectivity of cAMP against other cyclic nucleotides and cooperativity in ligand binding are still not known. Natural occurring CRP mutants with different phenotypes were employed to address these issues. Thermodynamic analyses of subunit association, protein stability, and cAMP and DNA binding as well as conformational studies of the mutants and wild-type CRPs lead to an identification of the apparently dominant roles of residues 128 and 141 in the cAMP-modulated DNA binding activity of CRP. Serine 128 and the C-helix were implicated as playing a critical role in modulating negative cooperativity of cyclic nucleotide binding. A correlation was established between a weak affinity for subunit assembly and the relaxation of cyclic nucleotide selectivity in the G141Q and S128A/G141Q mutants. These results imply that intersubunit interaction is important for cyclic nucleotide discrimination in CRP. The double mutant S128A/G141Q, constructed from two single mutations of S128A and G141Q, which exhibit opposite phenotypic characteristics of CRP- and CRP*, respectively, assumes a CRP* phenotype and has biochemical properties similar to those of the G141Q mutant. These observations suggest that mutation G141Q exerts a dominant effect over mutation S128A and that the subunit realignment induced by the G141Q mutation can override the local structural disruption created by mutation S128A.

Effects of single-residue substitutions on negative cooperativity in ligand binding to dihydrofolate reductase.

The effects of six amino acid substitutions in Lactobacillus casei dihydrofolate reductase, predominantly in the coenzyme binding site, on catalysis and on the negative cooperativity between NADPH and tetrahydrofolate binding have been determined. Replacement of Leu62, His64 or Leu54 by alanine has no effect on kcat, and produces only modest changes in negative cooperativity. Alanine substitution of His77, which interacts indirectly with the coenzyme adenine ring, leads to a doubling of the negative cooperativity and a consequent doubling of kcat. Replacement of Arg43, which interacts with the coenzyme 2'-phosphate, by alanine, or of Trp21, which interacts with the coenzyme nicotinamide ring, by histidine leads to a 20-100-fold decrease in negative cooperativity. In both mutants there is a decrease in kcat; isotope effects show that product release is largely rate-limiting in R43A, whereas in W21H hydride ion transfer is rate-limiting. 1H NMR has been used to obtain information on the extent of the structural changes produced by the substitutions. This varies from very local effects in H64A to very widespread effects in W21H. These changes are used as the basis for discussion of the mechanisms of the functional effects of the various substitutions. It is suggested that residues in helix C, beta-strand 3 and the beta3-beta4 loop may be involved in the transmission of effects between the coenzyme and substrate binding sites.

Analysis of the Binding of Ligands to Large Numbers of Sites: The Binding of Tryptophan to the 11 Sites of thetrpRNA-Binding Attenuation Protein

Analysis of the cooperative binding of ligands may provide insight into the underlying mechanisms of biological control. Binding ofl-tryptophan at each juncture in the ring of 11 subunits of thetrpRNA-binding attenuation protein exhibits cooperativity. To analyze binding of this kind we have developed both the matrix and combinatorial procedures for binding to large numbers of sites. A new, exact, and fast combinatorial procedure is presented. In this procedure the sites may be single or overlapping in a linear lattice or in the form of a ring. The dimensions of the ring of 11 binding sites forl-tryptophan on thetrpRNA-binding attenuation protein are such as to prevent direct interactions between thel-tryptophan molecules. Reasonable and explicit assumptions on analyzing the binding data lead to the conclusion that binding ofl-tryptophan raises the energy level of (destabilizes) the 11 membered structure.

Cooperativity and Dimerization of Recombinant Human Estrogen Receptor Hormone-binding Domain

The estrogen receptor dimerizes and exhibits cooperative ligand binding as part of its normal functioning. Interaction of the estrogen receptor with its ligands is mediated by a C-terminal hormone-binding domain (HBD), and residues within the HBD are thought to contribute to dimerization. To examine dimer interactions in the isolated HBD, a human estrogen receptor HBD fragment was expressed in high yield as a cleavable fusion protein in Escherichia coli. The isolated HBD peptide exhibited affinity for estradiol, ligand discrimination, and cooperative estradiol binding (Hill coefficient approximately 1.6) similar to the full-length protein. Circular dichroism spectroscopy suggests that the HBD contains significant amounts of alpha-helix ( approximately 60%) and some beta-strand ( approximately 7%) and that ligand binding induces little change in secondary structure. HBD dimer dissociation, measured using size exclusion chromatography, exhibited a half-life of approximately 1.2 h, which ligand binding increased approximately 3-fold (estradiol) to approximately 4-fold (4-hydroxytamoxifen). These results suggest that the isolated estrogen receptor HBD dimerizes and undergoes conformational changes associated with cooperative ligand binding in a manner comparable to the full-length protein, and that one effect of ligand binding is to alter the receptor dimer dissociation kinetics.

Quantitative analysis of ligand-macromolecule interactions using differential dynamic quenching of the ligand fluorescence to monitor the binding

Quantitative analyses of the thermodynamics and kinetics of ligand-macromolecule interactions in biological systems rely predominately on monitoring changes in the spectroscopic properties of the ligand or macromolecule, particularly fluorescence changes, which accompany the formation of the studied complexes. However, in many instances the interactions do not affect the fluorescence properties of interacting species and do not provide a resolution high enough to perform quantitative and rigorous measurements of the thermodynamic and/or kinetic parameters. In this communication, we describe the theoretical and experimental aspects of a method of studying complex, multiple ligand-macromolecule interactions by the fluorescence titration technique, when the intrinsic fluorescence changes accompanying binding do not provide a resolution necessary to perform quantitative analyses. The method is based on the fact that a fluorescent ligand, or binding sites of the macromolecule, can have different accessibility to the collisional (dynamic) quencher, when involved in the complex, rather than in the free, unbound state. The presence of an external dynamic quencher in solution, i.e., the presence of an extra collisional quenching process, transforms the fluorescence of the ligand or macromolecule, intrinsically independent of the complex formation, into a property which is dramatically different in the free state than in the bound state of the fluorophore. The approach is applicable to any model of noncooperative or cooperative ligand binding to a macromolecule and allows for the optimization of the resolution of the binding or kinetic studies for a given ligand-macromolecule system. The application of the method is illustrated by applying it to the study of the binding of the fluorescent derivative of a nucleotide cofactor, εADP, to the six interacting sites of the E. coli primary replicative helicase DnaB protein hexamer.

The peptide "FRCRCFa", dialysed intracellularly, inhibits the Na/Ca exchange in rabbit ventricular myocytes with high affinity.

We investigated the effect in rabbit ventricular myocytes of "FRCRCFa", a newly developed peptide inhibitor of the Na/Ca exchange. Myocytes were whole-cell patch clamped and experiments were carried out at 36 degrees C. The Na/Ca exchange was measured selectively, by blocking interfering ion channel currents and the Na/K pump, as the membrane current which could be inhibited by 5 mM nickel (Ni; a known blocker of the Na/Ca exchange). Increasing concentrations of FRCRCFa dialysed into the cell from the patch-pipette inhibited the Na/Ca exchange current. The dose/response curve could be fitted by a function for co-operative ligand binding, which predicted a KD for FRCRCFa-mediated inhibition of 22.7 +/- 3.7 nM, with a Hill coefficient of 0.61 +/- 0.06. Pipette FRCRCFa concentrations of 1 micro;M and above were sufficient to cause complete inhibition of Na/Ca exchange current. The inhibitory effect of FRCRCFa was independent of membrane potential and relatively selective: 10 micro;M FRCRCFa dialysed into the cell had no effect on the L-type Ca current and delayed rectifier and inward rectifier K currents. Thus FRCRCFa appears to be a potent and relatively selective inhibitor of the Na/Ca exchange in intact cardiac myocytes, and may be of value for studies of the Na/Ca exchange.

A test of the role of the proximal histidines in the Perutz model for cooperativity in haemoglobin.

Human haemoglobin has long been a paradigm for cooperative ligand binding and allostery. Through analysis of the crystal structures of deoxyhaemoglobin and liganded haemoglobin, Perutz proposed a model for cooperativity in which the bond between the proximal histidine and the protein couples haem rearrangements to protein structure rearrangements. Here we test this model by deleting the bonds between the histidine imidazole side chain and the polypeptide. This detachment method allows us to determine directly the contribution of proximal histidine coupling to cooperativity of distal ligand binding. Proximal detachment significantly increases ligand affinity, reduces cooperativity, and prevents quaternary structure switching, in accord with the Perutz model. Residual cooperativity indicates that additional haem communication pathways exist that do not involve the proximal histidine coupling mechanism.

Hb Bruxelles, Deletion of Pheβ42, Shows a Low Oxygen Affinity and Low Cooperativity of Ligand Binding

Functional studies of partially purified hemoglobin (Hb) Bruxelles, Phebeta42 (CD1) --> 0 indicate a major shift in the allosteric equilibrium toward the deoxy (T state) conformation. While Hb A shows a roughly symmetrical oxygenation curve with maximum cooperativity near half-saturation, Hb Bruxelles shows mainly properties of the low affinity (T state) form. The oxygen equilibrium curves for purified (>80%) Hb Bruxelles show little cooperativity and a P50 (without 2,3-diphosphoglycerate) about twice that of Hb A. The low cooperativity for Hb Bruxelles is partially compensated by an increase in oxygen affinity of the deoxy conformation and a lower 2,3-diphosphoglycerate effect. The beta chains of normal Hb have consecutive phenylalanine residues at positions 41 and 42. DNA sequencing studies of Hb Bruxelles showed a deletion of the codon TTT, which corresponds to residue Phe42. The CO rebinding kinetics after flash photolysis show mainly the slow phase, characteristic of CO binding to the deoxy conformation. In phosphate buffer at pH 7, the slow phase dominates even at low photolysis levels, where the main reaction is ligand binding to the triply liganded form. This indicates a switchover point, from the deoxy to oxy conformation, occurring beyond three ligands for Hb Bruxelles. There are few natural mutants that show a change in the oxygen affinity and cooperativity as large as that observed for Hb Bruxelles.

Optimal design: A computer program to study the best possible spacing of design points for model discrimination

Optimal design is largely concerned with selecting design points so as to maximise the precision of parameter estimates once the correct model has been identified. Often, however, a priority is that the correct model has first to be identified from a set of candidate models, and this procedure requires a different set of design criteria. We suppose that a decision has to be made on statistical grounds between accepting either a correct model g 2( x, Θ) or a deficient model g 1( x, Φ), using goodness of fit criteria, where weighting is dictated by a weighting function w( x) and spacing is specified by a spacing function ∝( x). We describe, for the first time, the choice of spacing function required to generate points which are uniformly spaced with respect to the y axis (Uniform- Y design). Then we introduce appropriate norms S n ( Θ), Q( Θ) and R( n), to quantify the effects of alternative choices of spacing, density and number of design points on the probability of correct model identification. Typical problems of this general type in biochemistry, for example, would be determining the correct number of classes of receptors in a ligand binding experiment, or fixing the number of exponential components in a pharmacokinetic experiment. A computer program which performs the necessary calculations for these and similar problems is described, and illustrated by analysing some typical test cases. Results from our extended investigations are also summarised, leading to more general conclusions as to the best spacing and density of design points for optimal model discrimination. We also prove the remarkable and previously unsuspected fact that, in differentiating two site cooperative ligand binding from one site, the two best designs give identical parameter estimates for the deficient model but are not actually equivalent from the point of view of model discrimination.

Stereochemical requirements of chitin synthase for ligand binding at the allosteric site for N-acetylglucosamine.

The substrate kinetics of chitin synthase (CS) were non-Michaelian, irrespective of the type of enzyme preparation studied (105-S chitosomal CS, and 16-S ex walls), even in the presence of saturating GlcNAc. An unexplained idiosyncrasy of this enzyme, which is likely to be responsible for this phenomenon, is evident from the striking non-linearity of product deposition with time at low substrate or low enzyme concentrations, particularly in the absence of GlcNac. The possibility can be excluded that this non-linearity is due to the formation of soluble by-products or intermediates in the form of chito-oligomers, as shown by HPLC/pulsed amperometric detection analysis. Additional evidence was sought for the tenet that CS is homotropically-heterotropically regulated, at least under steady-state reaction conditions. Substrate kinetic curves established from rate data for the linear reaction phase only were used for modelling. These could be reasonably well parameterised on the basis of the Monod mathematical model for co-operative ligand binding. Within a series of test compounds used to assess the stereochemical conditions of the allosteric site of CS for effector binding, N-acetylglucosaminono-1,5-lactone oxime excelled. Requirements for effector binding are as follows: (a) an aminoglucopyranose skeleton with the amino function acetylated, and (b) a single-bonded oxo-function present at C(1), which is preferentially a hydrogen bond donor, that may be equatorially spaced off, but must not be alpha-anomeric. The implications of these findings for chitin synthesis in vivo are discussed in terms of a mechanistically based fitness of CS to operate efficiently under vastly different combinations of substrate could be coordinately linked to the catabolism of chitin.

Cooperative Oxygen Binding to Scapharca inaequivalvis Hemoglobin in the Crystal

Oxygen binding to homodimeric Scapharca inaequivalvis hemoglobin (HbI) crystals has been investigated by single-crystal polarized absorption microspectrophotometry. The saturation curve, characterized by a Hill coefficient nH = 1.45 and an oxygen pressure at half saturation p50 = 4.8 torr, at 15 degrees C, shows that HbI in the crystalline state retains positive cooperativity in ligand binding. This finding will permit the correlation of the oxygen-linked conformational changes in the crystal with the expression of cooperativity. Polarized absorption spectra of deoxy-HbI, oxy-HbI, and oxidized HbI crystals indicate that oxygenation does not induce heme reorientation, whereas oxidation does. Lattice interactions prevent the dissociation of oxidized dimers that occurs in solution and stabilize an equilibrium distribution of pentacoordinate and hexacoordinate high spin species.

The cluster-arranged cooperative model: a model that accounts for the kinetics of binding to A1 adenosine receptors.

To explain the equilibrium binding and binding kinetics of ligands to membrane receptors, a number of models have been proposed, none of which is able to adequately describe the experimental findings, in particular the apparent negative cooperativity of ligand binding. In this paper, a new model, the cluster-arranged cooperative model, is presented whose main characteristic is that it explains the existence of negative cooperativity in the binding of ligands to the receptor molecule. The model is based on our findings of agonist binding to A1 adenosine receptors and of ligand-induced clustering of these receptors on the cell surface. The model assumes the existence of two conformational forms of the receptor in an equilibrium which depends on the concentration of the ligand. In this way, negative cooperativity is explained by the transmission of the information between receptor molecules through the structure of the membrane. The model is able to predict the thermodynamic binding and binding kinetics of [3H]-(R)-(phenylisopropyl)adenosine to A1 adenosine receptors in the presence and absence of guanylyl imidodiphosphate. In the presence of the guanine nucleotide analogue, the linear Scatchard plots obtained for [3H]-(R)-(phenylisopropyl)adenosine binding are explained by the disappearance of cooperativity, thus suggesting that G proteins are important for the existence of negative cooperativity in ligand binding. Among other predictions, the model justifies early events in homologous desensitization since high ligand concentrations would lead to the saturation of the receptor in a low-affinity conformation that does not signal. Our model can likely explain the behavior of a number of heptaspanning and tyrosine-kinase receptors exhibiting complex binding kinetics.

Cooperativity in ligand binding expressed at a model cell membrane by the vancomycin group antibiotics

Dimerisation or the use of a membrane anchor enhances the binding of the glycopeptide antibiotics at the surface of a model cell membrane.

Loss of allosteric behaviour in recombinant hemoglobin α2β29 r)F8)His→Ala: Restoration upon addition of strong effectors

In the stereochemical model proposed by Perutz [1], the Fe-His(F8) bond plays a significant role in the allosteric transition in hemoglobin and the resulting cooperativity in ligand binding. When this bond is ruptured, there is a loss in the transmission of the information concerning ligand binding; examples are Hb(NO) 4 in the presence of inositol hexakisphosphate (IHP), or nickel substituted Hb hybrids which, despite being liganded, exhibit deoxy-like properties. To study the effects of the loss of the iron proximal histidine bond, we have engineered the α 2 β 2(F8)H92A recombinant Hb. The replacement of the highly conserved proximal histidine F8 residue by an alanine results in a low affinity for the heme group and a loss of the allosteric properties; kinetics of CO recombination after photodissociation show only the rapid bimolecular phase, characteristic of the high affinity R-state. However, a significant amount of deoxy (T-state) kinetics are observed after addition of external effectors such as IHP. The iron-histidine bond is apparently crucial for the heme-heme interaction, but the allosteric equilibrium may still be influenced by external constraints.

Rapid Substrate Translocation by the Multisubunit, Erythroid Glucose Transporter Requires Subunit Associations but Not Cooperative Ligand Binding

The human erythroid glucose transporter is a GLUT1 homotetramer whose structure and function are stabilized by noncovalent, cooperative subunit interactions. The present study demonstrates that exofacial tryptic digestion of GLUT1 abolishes cooperative interactions between substrate binding sites on adjacent subunits under circumstances where subunit associations and high catalytic turnover are maintained. Extracellular trypsin produces rapid, quantitative cleavage of the human red cell-resident sugar transport protein, GLUT1. One major carboxyl-terminal peptide of M(r)(app) 25,000 is detected by immunoblot analysis. Endofacial tryptic digestion of GLUT1 results in the complete loss of GLUT1 carboxyl-terminal structure. GLUT1-mediated erythrocyte sugar uptake, transport inhibition by cytochalasin B, and GLUT1 oligomeric structure are unaffected by exofacial GLUT1 proteolysis. In contrast, the cytochalasin B binding capacity of GLUT1 and the Kd(app) for cytochalasin B binding to the transporter are doubled following exofacial tryptic digestion of GLUT1. Photoaffinity labeling experiments show that increased cytochalasin B binding results from increased ligand binding to the 25 kDa carboxyl-terminal GLUT1 peptide. Proteolysis abolishes allosteric interactions between sugar import (maltose binding) and sugar export (cytochalasin B binding) sites that normally exist on adjacent subunits within the transporter complex, but interact with negative cooperativity. Following exofacial proteolysis, these sites become mutually exclusive. Dithiothreitol disrupts GLUT1 quaternary structure, inhibits 3-O-methylglucose transport, and abolishes cooperative interactions between sugar import and export sites in control cells. Studies with reconstituted purified GLUT1 confirm that the action of trypsin on cytochalasin B binding is direct, show that proteolysis increases the apparent affinity of the sugar efflux site for transported sugars, and suggest that the membrane bilayer stabilizes GLUT1 noncovalent structure and catalytic function following GLUT1 proteolysis. Collectively, these findings demonstrate that GLUT1 does not require an intact polypeptide backbone for catalytic function. They show that the multisite sugar transporter mechanism is converted to a simple ping-pong carrier mechanism following exofacial GLUT1 proteolysis. They reveal that subunit cooperativity can be lost under circumstances where cohesive structural interactions between transporter subunits are maintained. They also refute the hypothesis [Hebert, D. N., & Carruthers, A. (1992) J. Biol. Chem. 267, 23829-23838] that rapid substrate translocation by the multisubunit erythroid glucose transporter requires cooperative interactions between subunit ligand binding sites.