Mechanism Of Allosteric Regulation Research Articles

Nonaggregating mutant of recombinant human hexokinase I exhibits wild-type kinetics and rod-like conformations in solution.

Hexokinase I governs the rate-limiting step of glycolysis in brain tissue, being inhibited by its product, glucose 6-phosphate, and allosterically relieved of product inhibition by phosphate. On the basis of small-angle X-ray scattering, the wild-type enzyme is a monomer in the presence of glucose and phosphate at protein concentrations up to 10 mg/mL, but in the presence of glucose 6-phosphate, is a dimer down to protein concentrations as low as 1 mg/mL. A mutant form of hexokinase I, specifically engineered by directed mutation to block dimerization, remains monomeric at high protein concentration under all conditions of ligation. This nondimerizing mutant exhibits wild-type activity, potent inhibition by glucose 6-phosphate, and phosphate reversal of product inhibition. Small-angle X-ray scattering data from the mutant hexokinase I in the presence of glucose/phosphate, glucose/glucose 6-phosphate, and glucose/ADP/Mg2+/AlF3 are consistent with a rodlike conformation for the monomer similar to that observed in crystal structures of the hexokinase I dimer. Hence, any mechanism for allosteric regulation of hexokinase I should maintain a global conformation of the polypeptide similar to that observed in crystallographic structures.

Accelerated Transcription of PRPS1 in X-linked Overactivity of Normal Human Phosphoribosylpyrophosphate Synthetase

Phosphoribosylpyrophosphate (PRPP) synthetase (PRS) superactivity is an X-linked disorder characterized by gout with overproduction of purine nucleotides and uric acid. Study of the two X-linked PRS isoforms (PRS1 and PRS2) in cells from certain affected individuals has shown selectively increased concentrations of structurally normal PRS1 transcript and isoform, suggesting that this form of the disorder involves pretranslational dysregulation of PRPS1 expression and might be more appropriately termed overactivity of normal PRS. We applied Southern and Northern blot analyses and slot blotting of nuclear runoffs to delineate the process underlying aberrant PRPS1 expression in fibroblasts and lymphoblasts from patients with overactivity of normal PRS. Neither PRPS1 amplification nor altered stability or processing of PRS1 mRNA was identified, but PRPS1 transcription was increased relative to GAPDH (3- to 4-fold normal in fibroblasts; 1.9- to 2.4-fold in lymphoblasts) and PRPS2. Nearly coordinate relative increases in each process mediating transfer of genetic information from PRPS1 transcription to maximal PRS1 isoform expression in patient fibroblasts further supported the idea that accelerated PRPS1 transcription is the major aberration leading to PRS1 overexpression. In addition, modulated relative increases in PRS activities at suboptimal Pi concentration and in rates of PRPP and purine nucleotide synthesis in intact patient fibroblasts indicate that despite an intact allosteric mechanism of regulation of PRS activity, PRPS1 transcription is a major determinant of PRPP and purine synthesis. The genetic basis of disordered PRPS1 transcription remains unresolved; normal- and patient-derived PRPS1s share nucleotide sequence identity at least 850 base pairs 5' to the consensus transcription initiation site.

Quantum mechanical analysis of oxygenated and deoxygenated states of hemocyanin: theoretical clues for a plausible allosteric model of oxygen binding.

In this work with ab initio computations, we describe relevant interactions between protein active sites and ligands, using as a test case arthropod hemocyanins. A computational analysis of models corresponding to the oxygenated and deoxygenated forms of the hemocyanin active site is performed using the Density Functional Theory approach. We characterize the electron density distribution of the binding site with and without bound oxygen in relation to the geometry, which stems out of the crystals of three hemocyanin proteins, namely the oxygenated form from the horseshoe crab Limulus polyphemus, and the deoxygenated forms, respectively, from the same source and from another arthropod, the spiny lobster Panulirus interruplus. Comparison of the three available crystals indicate structural differences at the oxygen binding site, which cannot be explained only by the presence and absence of the oxygen ligand, since the geometry of the ligand site of the deoxygenated Panulirus hemocyanin is rather similar to that of the oxygenated Limulus protein. This finding was interpreted in the frame of a mechanism of allosteric regulation for oxygen binding. However, the cooperative mechanism, which is experimentally well documented, is only partially supported by crystallographic data, since no oxygenated crystal of Panulirus hemocyanin is presently available. We address the following question: is the local ligand geometry responsible for the difference of the dicopper distance observed in the two deoxygenated forms of hemocyanin or is it necessary to advocate the allosteric regulation of the active site conformations in order to reconcile the different crystal forms? We find that the difference of the dicopper distance between the two deoxygenated hemocyanins is not due to the small differences of ligand geometry found in the crystals and conclude that it must be therefore stabilized by the whole protein tertiary structure.

Mechanism of Allosteric Regulation of the Rod cGMP Phosphodiesterase Activity by the Helical Domain of Transducin α Subunit

The G protein alpha subunit (Galpha) is composed of two distinct folding domains: a GTP-binding Ras-like domain and an alpha helical domain (HD). We have recently reported that the helical domain (HDt) of the vertebrate visual transducin alpha subunit (Galphat) synergizes activation of retinal cyclic GMP phosphodiesterase (PDE) by activated Galphat (Liu, W., and Northup, J. K., (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12878-12883). Here, we examine the molecular basis for this HD-based signaling regulation, and we provide a new model for the activation of the target effector. The HD proteins derived from visual transducin or taste gustducin alpha subunits, but no other Galpha HD proteins, each attenuate the PDE catalytic core (Palphabeta) and synergize Galphat stimulation of the holoPDE (Palphabetagamma2) with similar apparent affinities. The data from studies of both HDt-mediated attenuation and stimulation indicate that the HDt and the PDE inhibitory subunit (Pgamma) interact with PDE at independent sites and that Palphabeta contains the binding sites for HD. The saturation of both processes by HDt displays positive cooperativity with Hill coefficients of 1.5 for the attenuation of Palphabeta activity and 2.1 for synergism of holoPDE activation. Our data suggest the that Galphat-HDt regulates PDE by allosterically decreasing the affinity of Palphabeta for Pgamma and thus simultaneously facilitating the interaction of the activated Galphat-Ras-like domain with Pgamma. Thus, we propose a new model for the high efficiency of PDE activation as well as deactivation, and, overall, a novel mechanism for controlling fidelity, sensitivity, and efficacy of G protein signaling.

Role of a dynamic loop in cation activation and allosteric regulation of recombinant porcine fructose-1,6-bisphosphatase.

A disordered loop (loop 52-72, residues 52-72) in crystal structures of fructose-1,6-bisphosphatase (FBPase) has been implicated in regulatory and catalytic phenomena by studies in directed mutation. A crystal structure of FBPase in a complex with three zinc cations and the products fructose 6-phosphate (F6P) and phosphate (Pi) reveals loop 52-72 for the first time in a well-defined conformation with strong electron density. Loop 52-57 interacts primarily with the active site of its own subunit. Asp68 of the loop hydrogen bonds with Arg276 and a zinc cation located at the putative potassium activation site. Leu56 and Tyr57 of the loop pack against hydrophobic residues from two separate subunits of FBPase. A mechanism of allosteric regulation of catalysis is presented, in which AMP, by binding to its allosteric pocket, displaces loop 52-72 from the active site. Furthermore, the current structure suggests that both the alpha- and beta-anomers of F6P can be substrates in the reverse reaction catalyzed by FBPase. Mechanisms of catalysis are proposed for the reverse reaction in which Asp121 serves as a catalytic base for the alpha-anomer and Glu280 serves as a catalytic base for the beta-anomer.

Studies of the allosteric properties of maize leaf phosphoenolpyruvate carboxylase with the phosphoenolpyruvate analog phosphomycin as activator

The antibiotic phosphomycin (1,2-epoxypropylphosphonic acid), an analog of phosphoenolpyruvate (PEP), behaved not as an inhibitor, but as an activator, of the enzyme phosphoenolpyruvate carboxylase (PEPC) from maize leaves. Multiple activation studies indicated that the analog binds to the Glc6P-allosteric site producing a more activated enzyme than Glc6P itself. Because of this, we used phosphomycin as a tool to further extend our understanding of the mechanisms of allosteric regulation of C 4-PEPC. Initial velocity data from detailed kinetic studies, in which the concentrations of free and Mg-complexed PEP and phosphomycin were controlled, are consistent with: (1) the true activator is free phosphomycin, which competes with free PEP for the Glc6P-allosteric site; and (2) the Mg-phosphomycin complex caused inhibition by binding to the active site in competition with MgPEP. Therefore, although the Glc6P-allosteric site and the active site are able to bind the same ligands, they differ in the form of substrate and activator they bind. This important difference allows the full expression of the potential of activation and prevents inhibition by the activators, including the physiological ones, which are mostly uncomplexed at physiological free Mg 2+ concentrations. At fixed low substrate concentrations, the saturation kinetics of the enzyme by phosphomycin showed positive cooperativity at pH 7.3 and 8.3, although at the latter pH, the kinetics of saturation by the substrate was hyperbolic. The cosolute glycerol greatly increased the affinity of the enzyme for phosphomycin and abolished the cooperativity in its binding, but did not eliminate the heterotropic effects of the activator. Therefore, the heterotropic and homotropic effects of the activator are not always coupled to the homotropic effects of the substrate, which argues against the two-state model previously proposed to explain the allosteric properties of maize-leaf PEPC.

Interfacial communications in recombinant rabbit kidney pyruvate kinase.

Tissue-specific isozymes of pyruvate kinase are particularly attractive systems to elucidate the molecular mechanism(s) of conferring allostery. The muscle- and kidney-type isozymes are coded by the same gene. As a consequence of alternative message RNA splicing, the two primary sequences differ by a small number of residues. However, they exhibit very different regulatory behavior. In an effort to identify the roles of specific residues in conferring allostery, the gene encoding rabbit kidney-type pyruvate kinase was cloned and expressed in Escherichia coli. The primary structure of recombinant rabbit kidney-type pyruvate kinase (rRKPK) and recombinant rabbit muscle-type pyruvate kinase (rRMPK) differ at 22 positions, which are located in a region that forms important intersubunit contacts in the RMPK structure. Velocity sedimentation and analytical gel chromatographic studies show that rRKPK undergoes reversible dimer left and right arrow tetramer assembly with an equilibrium constant of 28 +/- 3 mL/mg. This subunit assembly process provides the opportunity to elucidate the role of this dimer interface in transmission of signal upon binding of substrates and allosteric effectors. The assembly to tetrameric rRKPK is favored by the binding of phosphoenolpyruvate (PEP), one of the two substrates, or fructose 1,6-bisphosphate (FBP), an activator. In contrast, the equilibrium is shifted toward dimeric rRKPK upon binding of adenosine diphosphate (ADP), the other substrate, or l-phenylalanine (Phe), the inhibitor. These observations provide significant new insights to the molecular mechanism of allosteric regulation in the pyruvate kinase system. First, all substrates and effectors communicate through this particular dimer-dimer interface. Second, the thermodynamic signatures of these communications are qualitatively different for the two substrates and between the activator, FBP, and inhibitor, Phe.

The allosteric regulation of pyruvate kinase

Crystallographic and mutagenesis studies have unravelled the general features of the allosteric transition mechanism in pyruvate kinase. The enzyme displays a dramatic conformational change in going from the T- to the R-state. All three domains forming each subunit of the tetrameric enzyme undergo simultaneous and concerted rotations, in such a way that all subunit and domain interfaces are modified. This mechanism is unpreceDAnted since in all tetrameric allosteric enzymes, characterised at atomic resolution, at least one of the domain or subunit interfaces remains unchanged on the T- to R-state transition. The molecular mechanism of allosteric regulation here proposed proviDAs a rationale for the effect of single site mutations observed in the human erythrocyte pyruvate kinase associated with a congenital anaemia.

Effects of lipids on nucleotide inhibition of wheat-germ aspartate transcarbamoylase: evidence of an additional level of control?

Wheat-germ aspartate transcarbamoylase, a monofunctional trimer, is strongly inhibited by uridine 5'-monophosphate (UMP), which shows kinetic interactions with the substrate, carbamoyl phosphate, suggesting a classical allosteric mechanism of regulation. Inhibition of the purified enzyme by UMP was amplified in the presence of a variety of ionic lipids at concentrations low enough to preclude denaturation. In the absence of UMP, most of these compounds had no kinetic effect or were slightly activating. Two phospholipids did not show the effect. In a homologous series of fatty acids (C6-C16), the potentiating effect was only seen with homologues greater than C8, reaching a maximum at C12. The effect of dodecanoate (C12) on kinetic cooperativity (UMP as variable ligand) was studied. At each of several fixed concentrations of carbamoyl phosphate, dodecanoate had a pronounced effect on the half-saturating concentration of UMP, which was reduced by about half in every case, indicating substantially tighter binding of UMP. However, dodecanoate had relatively little effect on the kinetic Hill coefficient for the cooperativity of UMP. The possible metabolic significance of these effects is discussed.

Allosteric Modulation by Tertiary Structure in Mammalian Hemoglobins: INTRODUCTION OF THE FUNCTIONAL CHARACTERISTICS OF BOVINE HEMOGLOBIN INTO HUMAN HEMOGLOBIN BY FIVE AMINO ACID SUBSTITUTIONS

Bovine erythrocytes do not contain 2,3-diphosphoglycerate, the principal allosteric effector of human hemoglobin. Bovine hemoglobin has a lower oxygen affinity than human hemoglobin and is regulated by physiological concentrations of chloride (Fronticelli, C., Bucci, E., and Razynska, A. (1988) J. Mol. Biol. 202, 343-348). It has been proposed that the chloride regulation in bovine hemoglobin is introduced by particular amino acid residues located in the amino-terminal region of the A helix and in the E helix of the beta subunits (Fronticelli, C. (1990) Biophys. Chem. 37, 141-146). In accordance with this proposal we have constructed two mutant human hemoglobins, beta(V1M+H2deleted+T4I+P5A) and beta(V1M+H2deleted+T4I+P5A+A76K). These are the residues present at the proposed locations in bovine hemoglobin except for isoleucine at position 4. Oxygen binding studies demonstrate that these mutations have introduced into human hemoglobin the low oxygen affinity and chloride sensitivity of bovine hemoglobin and reveal the presence of a previously unrecognized allosteric mechanism of oxygen affinity regulation where all the interactions responsible for the lowered affinity and chloride binding appear to be confined to individual beta subunits.

Small peptides activate the latent sequence-specific DNA binding function of p53

Normal cells contain p53 protein in a latent state that can be activated for sequence-specific transcription by low levels of UV radiation without an increase in protein levels. Microinjection of cells with an antibody specific to the C-terminal negative regulatory domain can activate the function of p53 as a specific transcription factor in the absence of irradiation damage, suggesting that posttranslational modification of a negative regulatory domain in vivo is a rate-limiting step for p53 activation. Small peptides derived from the negative regulatory domain of p53 have been used as biochemical tools to distinguish between allosteric and steric mechanisms of negative regulation of p53 tetramer activity. Presented is the development of a highly specific peptide activation system that is consistent with an allosteric mechanism of negative regulation and that forms a precedent for the synthesis of novel low molecular mass modifiers of the p53 response.

Dissociation of enzyme oligomers: a mechanism for allosteric regulation.

Most enzymes exist as oligomers or polymers, and a significant subset of these (perhaps 15% of all enzymes) can reversibly dissociate and reassociate in response to an effector ligand. Such a change in subunit assembly usually is accompanied by a change in enzyme activity, providing a mechanism for regulation. Two models are described for a physical mechanism, leading to a change in activity: (1) catalytic activity depends on subunit conformation, which is modulated by subunit dissociation; and (2) catalytic or regulatory sites are located at subunit interfaces and are disrupted by subunit dissociation. Examples of such enzymes show that both catalytic sites and regulatory sites occur at the junction of 2 subunits. In addition, for 9 enzymes, kinetic studies supported the existence of a separate regulatory site with significantly different affinity for the binding of either a substrate or a product of that enzyme. Over 40 dissociating enzymes are described from 3 major metabolic areas: carbohydrate metabolism, nucleotide metabolism, and amino acid metabolism. Important variables that influence enzyme dissociation include: enzyme concentration, ligand concentration, other cellular proteins, pH, and temperature. All these variables can be readily manipulated in vitro, but normally only the first two are physiological variables. Seven of these enzymes are most active as the dissociated monomer, the others as oligomers, emphasizing the importance of a regulated equilibrium between 2 or more conformational states. Experiments to test whether enzyme dissociation occurs in vivo showed this to be the case in 6 out of 7 studies, with 4 different enzymes.

Modulation of enzyme activity by antibody binding to an alkaline phosphatase-epitope hybrid protein.

An epitope from the HIV-1 gp120 protein V3 loop has been inserted onto the surface of bacterial alkaline phosphatase at different positions in the vicinity of the enzyme active site, creating hybrid proteins that can bind to an anti-gp120 monoclonal antibody. One of the hybrid proteins, API1, has a 13 amino acid V3 loop sequence inserted between residues 407 and 408 of alkaline phosphatase. The enzymatic activity of this protein is modulated upon antibody binding. API1 maintains the full activity of the wild type alkaline phosphatase but in the presence of the anti-gp120 antibody, the enzyme activity is inhibited by 40-50%. Thus, the hybrid enzyme can be used to detect the presence of antibody in solution. The concept of signalling proteins may have a wide application. Two models for the mechanism of modulation, steric hindrance and allosteric regulation, are discussed.

Hippocampal neurons exhibit cyclothiazide-sensitive rapidly desensitizing responses to kainate

In whole-cell recordings from mammalian CNS neurons, AMPA-preferring glutamate receptors exhibit strong desensitization in response to AMPA, glutamate, and quisqualate, but not to kainate or domoate. Such desensitization is reduced by lectins, by the nootropic drug aniracetam, and by diazoxide. None of these compounds strongly modulate responses to kainate and domoate, consistent with the apparent lack of desensitization to these agonists. We now report experiments on hippocampal neurons in which responses to kainate were strongly potentiated by cyclothiazide, a benzothiadiazine diuretic and antihypertensive drug structurally related to diazoxide. Cyclothiazide increased the maximum response to a saturating concentration of kainate by approximately 300% and produced a shift to the left in the kainate dose-response curve. Because cyclothiazide was considerably more effective than aniracetam in reducing desensitization evoked by glutamate, we tested the possibility that potentiation of responses to kainate was due to block of a previously undetected component of desensitization in the response to kainate itself. In outside-out patches responses to rapid perfusion of 3 mM kainate showed 34% desensitization, the onset of which developed with a time constant of 2.2 msec. Desensitization of responses to kainate was abolished by 100 microM cyclothiazide, as was the much stronger desensitization evoked by glutamate and AMPA. Cyclothiazide also slowed the rate of deactivation of responses to kainate recorded after return to agonist-free solution. Current-voltage plots for control responses to kainate exhibited outward rectification that was associated with a reduction in the amount of desensitization on depolarization. Both effects were absent in the presence of cyclothiazide, suggesting that rectification of responses to kainate was due to the voltage dependence of desensitization. The complete block of desensitization produced by cyclothiazide provides a powerful new tool for analysis of allosteric regulatory mechanisms at AMPA-preferring glutamate receptors.

Crystal structure of deoxygenated limulus polyphemus subunit II hemocyanin at 2.18 Å resolution: Clues for a mechanism for allosteric regulation

The crystal structure of Limulus polyphemus subunit type II hemocyanin in the deoxygenated state has been determined to a resolution of 2.18 A. Phase information for this first structure of a cheliceratan hemocyanin was obtained by molecular replacement using the crustacean hemocyanin structure of Panulirus interruptus. The most striking observation in the Limulus structure is the unexpectedly large distance of 4.6 A between both copper ions in the oxygen-binding site. Each copper has approximate trigonal planar coordination by three histidine N epsilon atoms. No bridging ligand between the copper ions could be detected. Other important new discoveries are (1) the presence of a cis-peptide bond between Glu 309 and Ser 310, with the carbonyl oxygen of the peptide plane hydrogen bonded to the N delta atom of the copper B ligand His 324; (2) localization of a chloride-binding site in the interface between the first and second domain; (3) localization of a putative calcium-binding site in the third domain. Furthermore, comparison of Limulus versus Panulirus hemocyanin revealed considerable tertiary and quaternary rigid body movements, although the overall folds are similar. Within the subunit, the first domain is rotated by about 7.5 degrees with respect to the other two domains, whereas within the hexamer the major movement is a 3.1 degrees rotation of the trimers with respect to each other. The rigid body rotation of the first domain suggests a structural mechanism for the allosteric regulation by chloride ions and probably causes the cooperative transition of the hexamer between low and high oxygen affinity states. In this postulated mechanism, the fully conserved Phe49 is the key residue that couples conformational changes of the dinuclear copper site into movements of the first domain.

Homoglobin Rouen ( α-140 (HC2) Tyr→His): alteration of the α-chain C-terminal region and moderate increase in oxygen affinity

Hb Rouen ( α140 (HC2) Tyr→His) is a moderately high oxygen-affinity variant that was found in coincidence with polycythemia vera in a French patient. This hemoglobin provides an example of an alteration of the C-terminus of the α-chain, a region involved in the mechanisms of allosteric regulation. The increase in oxygen-affinity and decrease in cooperativity of this variant is much smaller than that resulting from the same substitution in the β-chain. This model provides additional evidence for the inequivalence between the α- and β-subunits.

Mechanisms of cooperativity and allosteric regulation in proteins. M. Perutz. Cambridge University Press: Cambridge, x + 101 pages, £11.95 (1990)

Mechanisms of cooperativity and allosteric regulation in proteins. M. Perutz. Cambridge University Press: Cambridge, x + 101 pages, £11.95 (1990)

Structural heterogeneity of membrane receptors and GTP-binding proteins and its functional consequences for signal transduction.

Recent information obtained, mainly by recombinant cDNA technology, on structural heterogeneity of hormone and transmitter receptors, of GTP-binding proteins (G-proteins) and, especially, of G-protein-linked receptors is reviewed and the implications of structural heterogeneity for diversity of hormone and transmitter actions is discussed. For the future, three-dimensional structural analysis of membrane proteins participating in signal transmission and transduction pathways is needed in order to understand the molecular basis of allosteric regulatory mechanisms governing the interactions between these proteins including hysteretic properties and cell-cybernetic aspects.

M. Perutz. Mechanisms of cooperativity and allosteric regulation in proteins. Cambridge University Press, Cambridge 1990. 101 pp + x

Journal of Molecular RecognitionVolume 4, Issue 2-3 p. 111-111 Book Review M. Perutz. Mechanisms of cooperativity and allosteric regulation in proteins. Cambridge University Press, Cambridge 1990. 101 pp + x Edward Eisenstein, Edward Eisenstein Center for Advanced Research in Biotechnology, Rockville, MD USASearch for more papers by this author Edward Eisenstein, Edward Eisenstein Center for Advanced Research in Biotechnology, Rockville, MD USASearch for more papers by this author First published: March ‐ June 1991 https://doi.org/10.1002/jmr.300040210AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume4, Issue2-3March ‐ June 1991Pages 111-111 RelatedInformation

Mechanisms of cooperativity and allosteric regulation in proteins.

AUosteric proteins control and coordinate chemical events in the living cell. When Monod conceived that idea he said that he had discovered the second secret of life. The first was the structure of DNA. The theory as published by Monodet al.(1963) was concerned chiefly with cooperativity and feedback inhibition of enzymes, such as the inhibition of threonine deaminase, the first enzyme in the pathway of the synthesis of isoleucine, by isoleucine, and its activation by valine. Two years later the theory was formalized by Monodet al.(1965).