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PubMed2001

Kinetic and physiological effects of alterations in homologous isocitrate-binding sites of yeast NAD(+)-specific isocitrate dehydrogenase.

Yeast NAD(+)-specific isocitrate dehydrogenase is an allosterically regulated octameric enzyme composed of four each of two homologous but nonidentical subunits designated IDH1 and IDH2. Models based on the crystallographic structure of Escherichia coli isocitrate dehydrogenase suggest that both yeast subunits contain isocitrate-binding sites. Identities in nine residue positions are predicted for the IDH2 site whereas four of the nine positions differ between the IDH1 and bacterial enzyme sites. Thus, we speculate that the IDH2 site is catalytic and that the IDH1 site may bind but not catalytically alter isocitrate. This was examined by kinetic analyses of enzymes with independent and concerted replacement of residues in each yeast IDH subunit site with the residues that differ in the other subunit site. Mutant enzymes were expressed in a yeast strain containing disrupted IDH1 and IDH2 loci and affinity-purified for kinetic analyses. The primary effects of various residue replacements in IDH2 were reductions of 30->300-fold in V(max) values, consistent with the catalytic function of this subunit. In contrast, replacement of all four residues in IDH1 produced a 17-fold reduction in V(max) under the same assay conditions, suggesting that the IDH1 site is not the primary catalytic site. However, single or multiple residue replacements in IDH1 uniformly increased half-saturation concentrations for isocitrate, implying that isocitrate can be bound at this site. Both subunits appear to contribute to cooperativity with respect to isocitrate, but AMP activation is lost only with residue replacements in IDH1. Overall, results are consistent with isocitrate binding by IDH2 for catalysis and with isocitrate binding by IDH1 being a prerequisite for allosteric activation by AMP. The effects of residue substitutions on enzyme function in vivo were assessed by analysis of various growth phenotypes. Results indicate a positive correlation between the level of IDH catalytic activity and the ability of cells to grow with acetate or glycerol as carbon sources. In addition, lower levels of activity are associated with increased production of respiratory-deficient (petite) segregants.

Allosteric Regulation↗
PubMed1994

Crystal structure of fructose-1,6-bisphosphatase complexed with fructose 2,6-bisphosphate, AMP, and Zn2+ at 2.0-A resolution: aspects of synergism between inhibitors.

The crystal structure of fructose-1,6-bisphosphatase (Fru-1,6-Pase; EC 3.1.3.11) complexed with Zn2+ and two allosteric regulators, AMP and fructose 2,6-bisphosphate (Fru-2,6-P2) has been determined at 2.0-A resolution. In the refined model, the crystallographic R factor is 0.189 with rms deviations of 0.014 A and 2.8 degrees from ideal geometries for bond lengths and bond angles, respectively. A 15 degrees rotation is observed between the upper dimer C1C2 and the lower dimer C3C4 relative to the R-form structure (fructose 6-phosphate complex), consistent with that expected from a T-form structure. The major difference between the structure of the previously determined Fru-2,6-P2 complex (R form) and that of the current quaternary T-form complex lies in the active site domain. A zinc binding site distinct from the three binding sites established earlier was identified within each monomer. Helix H4 (residues 123-127) was found to be better defined than in previously studied ligated Fru-1,6-Pase structures. Interactions between monomers in the active site domain were found involving H4 residues from one monomer and residues Tyr-258 and Arg-243 from the adjacent monomer. Cooperativity between AMP and Fru-2,6-P2 in signal transmission probably involves the following features: an AMP site, the adjacent B3 strand (residues 113-118), the metal site, the immediate active site, the short helix H4 (residues 123-127), and Tyr-258 and Arg-243 from the adjacent monomer within the upper (or lower) dimer. The closest distance between the immediate active site and that on the adjacent monomer is only 5 A. Thus, the involvement of H4 in signal transmission adds another important pathway to the scheme of the allosteric mechanism of Fru-1,6-Pase.

Adenosine Monophosphate↗
PubMed1992

Key residues in the allosteric transition of Bacillus stearothermophilus pyruvate kinase identified by site-directed mutagenesis.

The structural gene for pyruvate kinase from Bacillus stearothermophilus has been cloned in Escherichia coli and sequenced. The open reading frame from the ATG start codon to the TAG stop codon is 1482 base-pairs and encodes a peptide of relative molecular mass 52,967. In the expression vector pKK223-3, containing the synthetic tac promoter, the gene is overexpressed in E. coli cells to an estimated level of 30% total soluble cell protein. A purification procedure for the overexpressed protein has been established. The construction and characterization of a pair of mutant proteins has given insight into the structural basis of allosteric regulation in the tetrameric enzyme. Substituting tryptophan for tyrosine at position 466 (mutant Trp466-->Tyr) resulted in an activated form of the enzyme, having a reduced K1/2 for the substrate phosphoenolpyruvate. We propose that the characteristics of this mutant might be the result of bulk removal releasing steric inhibition to the formation of an interdomain salt bridge between Asp356 and Arg444. The regulatory behaviour of the double mutant produced by making the additional substitution aspartate for glutamate at position 356 (Trp466-->Tyr/Asp356-->Glu) corroborates this. The position of the salt bridge is such that it might be pivotal to the conformation of a pocket that is proposed to open up when the active R-conformation is adopted. We suggest that the mechanism of activation of B. stearothermophilus pyruvate kinase by ribose-5-phosphate might hinge on an interaction with, or indirectly through, residue Trp466, removing it from the vicinity of the potential salt bridge between Asp356 and Arg444 and thus effecting a closing together of the protein structure concomitant with an opening up of the pocket region.

Allosteric Regulation↗
PubMed1996

Role of the active site gate of glycogen phosphorylase in allosteric inhibition and substrate binding.

The functional role in allosteric regulation of a flexible loop (residues 280-288) located near the active site of muscle glycogen phosphorylase was investigated. Mutations were made in residues 283-285 based on crystallographic studies that indicate that the loop functions as a gate controlling access of substrates to the active site and that these specific residues play distinct roles in mimicking the substrate and binding inhibitors when the enzyme is in an inactive conformation. Substitution of Ala or Asn for Asp-283, the putative substrate mimic, results in a 15-fold decrease in Vmax, a 10-fold decrease in the S0.5 for glucose 1-phosphate, a 10-fold increase in the Ka for AMP, and a 10-20-fold increase in the Ki for glucose. Substitution of Ala for Asn-284, which normally forms a hydrogen bond with the inhibitor glucose, reduces Vmax 3-fold, increases the Ki for glucose 2-fold, but has little effect on AMP or glucose 1-phosphate binding or cooperativity. Substitution of Asp at 284, on the other hand, reduces Vmax 10-fold, elevates the Ki for glucose 10-fold, decreases AMP cooperativity, but has little effect on the affinity of AMP or the cooperativity and binding of glucose 1-phosphate. Substitution of Leu for Phe-285, which forms aromatic stacking interactions with purine inhibitors, reduces Vmax 2-fold, decreases the affinity for caffeine at least 10-fold, raises the Ka for AMP 3-fold, and decreases AMP cooperativity but has little effect on glucose 1-phosphate binding or cooperativity. The results of the mutagenesis studies show the importance of the 280's loop for inhibitor binding and modulation of substrate affinity and suggest a role for the loop in allosteric activation. The propagation of allosteric effects across the domain interface may depend upon specific contacts between residues of the 280's loop and the C-terminal domain.

Adenosine Monophosphate↗
PubMed1998

Probing the regulatory domain interface of D-3-phosphoglycerate dehydrogenase with engineered tryptophan residues.

D-3-Phosphoglycerate dehydrogenase from Escherichia coli is a homotetrameric enzyme which is allosterically regulated by the end product of its pathway, L-serine. The enzyme binds 4 L-serine molecules at two interfaces formed by the noncovalent association of the regulatory domains. The two domains that comprise each interface are related by an approximately 180 degrees axis of symmetry, and two serine molecules bind at each interface by forming a hydrogen bond network between the domains. A model has been proposed that suggests that serine functions by drawing adjacent domains together and that this in turn translates a conformational change to the active site. A tryptophan residue has been engineered into the helices flanking the regulatory interfaces that displays significant quenching in response to serine binding. Residues on the adjacent subunit appear to be primarily responsible for the tryptophan quenching and thus support the hypothesis that serine binding leads to an increase in the proximity between residues on neighboring subunits. Serine binding studies show that this quenching, as well as inhibition of enzymatic activity, are essentially complete when only two of the four serine binding sites are occupied. The requirement for only one serine per interface is consistent with the notion that the interface is formed by relatively rigid domains and that hydrogen bonding at only a single site is all that is required to substantially close the interface. The fluorescence quenching in response to L-serine binding generally correlates with enzymatic inhibition, but there appears to be a slight lag in inhibition relative to quenching at low serine concentrations. The observed fluorescence quenching of residues in the regulatory domains of D-3-phosphoglycerate dehydrogenase provide the first direct evidence for a conformational change in response to effector binding and provide a means to monitor the first step in the allosteric mechanism.

Allosteric Regulation↗
PubMed1996

A study of subunit interface residues of fructose-1,6-bisphosphatase by site-directed mutagenesis: effects on AMP and Mg2+ affinities.

The structural transformation of fructose-1,6-bisphosphatase upon binding of the allosteric regulator AMP dramatically changes the interactions across the C1-C4 (C2-C3) subunit interface of the enzyme. Asn9, Met18, and Ser87 residues were modified by site-directed mutagenesis to probe the function of the interface residues in porcine liver fructose-1,6-bisphosphatase. The wild-type and mutant forms of the enzyme were purified to homogeneity and characterized by initial rate kinetics and circular dichroism (CD) spectrometry. No discernible alterations in structure were observed among the wild-type and Asn9Asp, Met18Ile, Met18Arg, and Ser87Ala mutant forms of the enzyme as measured by CD spectrometry. Kinetic analyses revealed 1.6- and 1.8-fold increases in kcat with Met18Arg and Asn9Asp, respectively. The K(m) for fructose 1,6-bisphosphate increased about 2-approximately 4-fold relative to that of the wild-type enzyme in the four mutants. A 50-fold lower Ka value for Mg2+ compared with that of the wild-type enzyme was obtained for Met18Ile with no alteration of the Ki for AMP. However, the replacement of Met18 with Arg caused a dramatic decrease in AMP affinity (20 000-fold) without a change in Mg2+ affinity. Increases of 6- and 2-fold in the Ki values for AMP were found with Asn9Asp and Ser87Ala, respectively. There was no difference in the cooperativity for AMP inhibition between the wild-type and the mutant forms of fructose-1,6-bisphosphatase. This study demonstrates that the mutation of residues in the C1-C4 (C2-C3) interface of fructose-1,6-bisphosphatase can significantly affect the affinity for Mg2+, which is presumably bound 30 A away. Moreover the mutations alternatively reduce AMP and Mg2+ affinities, and this finding may be associated with the destabilization of the corresponding allosteric states of the enzyme. The kinetics and structural modeling studies of the interface residues provide new insights into the conformational equilibrium of fructose-1,6-bisphosphatase.

Adenosine Monophosphate↗
PubMed2003

A metal-ligand-mediated intersubunit allosteric switch in related SmtB/ArsR zinc sensor proteins.

The origin of metal ion selectivity by members of the SmtB/ArsR family of bacterial metal-sensing transcriptional repressors and the mechanism of negative allosteric regulation of DNA binding is poorly understood. Here, we report that two homologous zinc sensors, Staphylococcus aureus CzrA and cyanobacterial SmtB, are "winged" helix homodimeric DNA-binding proteins that bind Zn(II) to a pair of tetrahedral, interhelical binding sites, with two ligands derived from the alpha5 helix of one subunit, Asp84 O(delta1) (Asp104 in SmtB), His86 N(delta1) (His106), and two derived from the alpha5 helix of the other, His97' N(delta1) (His117') and His100' N(epsilon2) (Glu120'). Formation of the metal chelate drives a quaternary structural switch mediated by an intersubunit hydrogen-binding network that originates with the non-liganding N(epsilon2) face of His97 in CzrA (His117 in SmtB) that stabilizes a low-affinity, DNA-binding conformation. The structure of the Zn(1) SmtB homodimer shows that both metal-binding sites of the dimer must be occupied for the quaternary structural switch to occur. Thus, a critical zinc-ligating histidine residue obligatorily couples formation of the metal-sensing coordination chelate to changes in the conformation and dynamics of the putative DNA-binding helices.

Allosteric Regulation↗
PubMed2002

Allosteric hammerhead ribozyme TRAPs.

A new mode of allosteric regulation of nucleic acid enzymes is described and shown to operate effectively with hammerhead ribozymes. In the "TRAP" design (for targeted ribozyme-attenuated probe), a 3' terminal "attenuator" anneals to conserved bases in the catalytic core to form the "off" state of the ribozyme. Binding of RNA or DNA to an antisense sequence linking the ribozyme and attenuator frees the core to fold into an active conformation, even though the antisense sequence itself does not interfere with the ribozyme. TRAP hammerheads based on the previously characterized HH8 ribozyme were shown to be activated more than 250-fold upon addition of the sense strand. RNA oligonucleotides were more effective activators than DNA oligos, consistent with the known relative helix stabilities (RNA-RNA > RNA-DNA). Oligonucleotides that directly paired with the attenuator gave up to 1760-fold activation. The magnitude of the activation was greater when the oligo was added prior to folding than if it was added during the cleavage reaction. The TRAP design requires no prior knowledge of (deoxy)ribozyme structure beyond identification of the essential core. Thus, this approach should be readily generalizable to other systems for biomedicine, sensor technology, and additional applications.

Allosteric Regulation↗
PubMed1999

An inducible gene product for 6-phosphofructo-2-kinase with an AU-rich instability element: role in tumor cell glycolysis and the Warburg effect.

Cancer cells maintain a high glycolytic rate even in the presence of oxygen, a phenomenon first described over 70 years ago and known historically as the Warburg effect. Fructose 2,6-bisphosphate is a powerful allosteric regulator of glycolysis that acts to stimulate the activity of 6-phosphofructo-1-kinase (PFK-1), the most important control point in mammalian glycolysis. The steady state concentration of fructose 2,6-bisphosphate in turn depends on the activity of the enzyme 6-phosphofructo-2-kinase (PFK-2)/fructose-2, 6-bisphosphatase, which is expressed in several tissue-specific isoforms. We report herein the identification of a gene product for this enzyme that is induced by proinflammatory stimuli and which is distinguished by the presence of multiple copies of the AUUUA mRNA instability motif in its 3'-untranslated end. This inducible gene for PFK-2 is expressed constitutively in several human cancer cell lines and was found to be required for tumor cell growth in vitro and in vivo. Inhibition of inducible PFK-2 protein expression decreased the intracellular level of 5-phosphoribosyl-1-pyrophosphate, a product of the pentose phosphate pathway and an important precursor for nucleic acid biosynthesis. These studies identify a regulatory isoenzyme that may be essential for tumor growth and provide an explanation for long-standing observations concerning the apparent coupling of enhanced glycolysis and cell proliferation.

3' Untranslated Regions↗
PubMed2004

Activation of integrin beta-subunit I-like domains by one-turn C-terminal alpha-helix deletions.

Integrins contain two structurally homologous but distantly related domains: an I-like domain that is present in all beta-subunits and an I domain that is present in some alpha-subunits. Atomic resolution and mutagenesis studies of alpha I domains demonstrate a C-terminal, axial displacement of the alpha7-helix that allosterically regulates the shape and affinity of the ligand-binding site. Atomic resolution studies of beta I-like domains have thus far demonstrated no similar alpha7-helix displacement; however, other studies are consistent with the idea that alpha I and beta I-like domains undergo structurally analogous rearrangements. To test the hypothesis that C-terminal, axial displacement of the alpha7-helix, coupled with beta6-alpha7 loop reshaping, activates beta I-like domains, we have mimicked the effect of alpha7-helix displacement on the beta6-alpha7 loop by shortening the alpha7-helix by two independent, four-residue deletions of about one turn of alpha-helix. In the case of integrin alphaLbeta2, each mutant exhibits constitutively high affinity for the physiological ligand intercellular adhesion molecule 1 and full exposure of a beta I-like domain activation-dependent antibody epitope. In the case of analogous mutants in integrin alpha4beta7, each mutant shows the activated phenotype of firm adhesion, rather than rolling adhesion, in shear flow. The results show that integrins that contain or lack alpha I domains share a common pathway of beta I-like domain activation, and they suggest that beta I-like and alpha I domain activation involves structurally analogous alpha7-helix axial displacements.

Allosteric Regulation↗
PubMed1998

Crystal structure of a complex of Escherichia coli glycerol kinase and an allosteric effector fructose 1,6-bisphosphate.

The three-dimensional structures of Escherichia coli glycerol kinase (GK) with bound glycerol in the presence and absence of one of the allosteric regulators of its activity, fructose 1,6-bisphosphate (FBP), at 3.2 and 3.0 A, are presented. The molecule crystallized in space group P41212, and the structure was solved by molecular replacement. The models were refined with good stereochemistry to final R-factors of 21.1 and 21.9%, respectively. A tetrameric arrangement of monomers was observed which was essentially identical to the proposed inactive tetramer II previously described [Feese, M. D., Faber, H. R., Bystrom, C. E., Pettigrew, D. W., and Remington, S. J. (1998) Structure (in press)]. However, the crystal packing in this form was especially open, permitting the FBP binding site to be occupied and identified. The crystallographic data revealed a most unusual type of FBP binding site formed between two glycine-arginine loops (residues 234-236) where one-half of the binding site is donated by each monomer at the regulatory interface. The molecule of FBP binds in two mutually exclusive modes on a noncrystallographic 2-fold axis at 50% occupancy each; thus, a tetramer of GK binds two molecules of FBP. Ionic interactions between the 1- and 6-phosphates of FBP and Arg 236 were observed in addition to hydrogen bonding interactions between the backbone amide of Gly 234 and the 6-phosphate. No contacts between the protein and the furanose ring were observed. Mutagenesis of Arg 236 to alanine drastically reduced the extent of inhibition of GK by FBP and lowered, but did not eliminate, the ability of FBP to promote tetramer association. These observations are consistent with the previously characterized mechanism of FBP inhibition of GK, in which FBP acts both to promote dimer-tetramer assembly and to inactivate the tetramers.

Alanine↗
PubMed1998

Conformational fluctuations in deoxy hemoglobin revealed as a major contributor to anionic modulation of function through studies of the oxygenation and oxidation of hemoglobins A0 and Deer Lodge beta2(NA2)His --> Arg.

Organisms rely on regulation at the molecular level, such as the allosteric regulation of hemoglobin (Hb) function by anions, to meet challenges presented by changing environmental and physiological conditions. A comparison of the effects of anions on oxygenation, oxidation, and sulfhydryl reactivity of Hb leads us to suggest that a large and significant part of the shift in oxygen affinity brought about by anion binding occurs as a result of increased conformational rigidity of the T state of deoxy Hb. As conformational rigidity increases, it becomes increasingly difficult for subunits in the deoxygenated T-state tetramer to assume higher oxygen affinity forms (T', T", T"'...) with less steric hindrance. The oxygen affinity reflects the average of the rapidly equilibrating conformations within the T state and is correspondingly decreased when anion levels are increased. The initial stage of the oxidation of Hb is relatively insensitive to steric alterations and thus reflects, primarily, the electronic aspects of the quaternary (T, T', T", T"'...) <--> equilibrium. We show that the reactivity of the sterically obscured sulfhydryl of beta93 Cys in deoxy Hb is much greater in chloride-free buffers than in buffers with added chloride. Anion-induced decreases in the extent and frequency of conformational fluctuations of subunits within the T-quaternary state thus reduce sulfhydryl reactivity as well as oxygen affinity. This parallel in anionic control of function allowed us to test, and disprove, the possibility that uncompensated positive charges in the central cavity of Hb Deer Lodge increase the frequency and extent of conformational fluctuations in its deoxy structure. This Hb variant exhibits increased anion sensitivity, increased oxygen affinity, and increased ease of oxidation, but without increased reactivity of its sulfhydryl groups, indicating that active-site alterations in deoxy Hb Deer Lodge are primarily electronic and not associated with increased conformational fluctuations within its T state. The restoration of normal properties in Hb Deer Lodge by addition of anions reinforces our conclusion that anionic control can be exerted through both steric and electronic alterations. The anionic control of fluctuations within the T state of Hb illustrates an important principle of macromolecular structure-function relationships: that functional regulation can be achieved by alterations in conformational rigidity.

Anions↗
PubMed1986

The association of NADPH with the guanine nucleotide exchange factor from rabbit reticulocytes: a role of pyridine dinucleotides in eukaryotic polypeptide chain initiation.

The guanine nucleotide exchange factor (GEF) was purified to apparent homogeneity from postribosomal supernatants of rabbit reticulocytes by chromatography on DEAE-cellulose and phosphocellulose, fractionation by glycerol gradients, and chromatography on Mono S and Mono Q (Pharmacia). At the Mono S step GEF is isolated as a complex with the eukaryotic polypeptide chain initiation factor 2 (eIF-2) and is separated from this factor by column chromatography on Mono Q. An emission spectrum characteristic of a reduced pyridine dinucleotide was observed when GEF was subjected to fluorescence analysis. By both coupled enzymatic analysis and chromatography on reverse-phase or Mono Q columns, the bound dinucleotide associated with GEF was determined to be NADPH. The GEF-catalyzed exchange of eIF-2-bound GDP for GTP was markedly inhibited by NAD+ and NADP+. This inhibition was not observed in the presence of equimolar concentrations of NADPH. Similarly, the stimulation of ternary complex (eIF-2 X GTP X Met-tRNAf) formation by GEF in the presence of 1 mM Mg2+ was abolished in the presence of oxidized pyridine dinucleotide. These results demonstrate that pyridine dinucleotides may be directly involved in the regulation of polypeptide chain initiation by acting as allosteric regulators of GEF activity.

Animals↗
PubMedDate not supplied

[Conformational stability of glutamate dehydrogenase and its complexes with specific ligands].

The isotermic denaturation of glutamatdehydrogenase (GDH) and its complexes with co-enzymes, substrates and allosteric regulators under the action of urea was studied. It was shown that the reaction of the enzyme with an allosteric inhibitor GTP is accompanied by a decrease in conformational stability of the catalytically active hexsamer GDH. Formation of a complex with the allosteric activator ADP increases the conformational stability of the enzyme. Studies on the isotermic unfolding of GDH in the presence of various phosphoric ethers of adenosine gave evidence that the stabilizing effect of ADP is based on the reaction of the enzyme with the adenine base of the regulator.

Adenosine Diphosphate↗
PubMed1997

Binding of allosteric effectors to ribonucleotide reductase protein R1: reduction of active-site cysteines promotes substrate binding.

BACKGROUND: Ribonucleotide reductase (RNR) is an essential enzyme in DNA synthesis, catalyzing all de novo synthesis of deoxyribonucleotides. The enzyme comprises two dimers, termed R1 and R2, and contains the redox active cysteine residues, Cys462 and Cys225. The reduction of ribonucleotides to deoxyribonucleotides involves the transfer of free radicals. The pathway for the radical has previously been suggested from crystallographic results, and is supported by site-directed mutagenesis studies. Most RNRs are allosterically regulated through two different nucleotide-binding sites: one site controls general activity and the other controls substrate specificity. Our aim has been to crystallographically demonstrate substrate binding and to locate the two effector-binding sites. RESULTS: We report here the first crystal structure of RNR R1 in a reduced form. The structure shows that upon reduction of the redox active cysteines, the sulfur atom of Cys462 becomes deeply buried. The more accessible Cys225 moves to the former position of Cys462 making room for the substrate. In addition, the structures of R1 in complexes with effector, effector analog and effector plus substrate provide information about these binding sites. The substrate GDP binds in a cleft between two domains with its beta-phosphate bound to the N termini of two helices; the ribose forms hydrogen bonds to conserved residues. Binding of dTTP at the allosteric substrate specificity site stabilizes three loops close to the dimer interface and the active site, whereas the general allosteric binding site is positioned far from the active site. CONCLUSIONS: Binding of substrate at the active site of the enzyme is structurally regulated in two ways: binding of the correct substrate is regulated by the binding of allosteric effectors and binding of the actual substrate occurs primarily when the active-site cysteines are reduced. One of the loops stabilized upon binding of dTTP participates in the formation of the substrate-binding site through direct interaction with the nucleotide base. The general allosteric effector site, located far from the active site, appears to regulate subunit interactions within the holoenzyme.

Allosteric Regulation↗
PubMed1994

Effect of aspartate and glutamate on the oxoglutarate carrier investigated in rat heart mitochondria and inverted submitochondrial vesicles.

Interaction of glutamate and aspartate with the oxoglutarate carrier was investigated in rat heart mitochondria or inverted submitochondrial particles. With mitochondria, glutamate and aspartate had no effect on the initial rate of oxoglutarate or malate uptake. With inverted submitochondrial vesicles, binding experiments indicated that aspartate bound to the oxoglutarate carrier on its matricial face and increased the affinity of the substrate binding site for malate but did not change the affinity for oxoglutarate. Glutamate had no effect on both substrate bindings. The dissociation constants of the binary substrate-carrier complexes on the matricial side were determined (1.28 +/- 0.15 mM for oxoglutarate and 2.22 +/- 0.26 mM for malate). These values, compared with those obtained previously on the cytosolic side of intact mitochondria, confirmed the asymmetry of the carrier in the native membrane (higher affinities on the cytosolic face). It is concluded that (1) aspartate and glutamate are not cytosolic effectors of the oxoglutarate carrier, (2) matricial aspartate is a positive effector of the binding of malate on the matricial side of the oxoglutarate carrier, and (3) such a characteristic may play a role in the regulation of the oxoglutarate carrier. Thus, it may be emphasized that (1) this observation is the first clear evidence of a well-defined 'sophisticated regulation' (allosteric) of a mitochondrial metabolite carrier, and (2) this regulation of the oxoglutarate carrier may have important consequences on the efficiency of reducing equivalent import in the matrix space by the malate-aspartate shuttle.

Animals↗
PubMed2001

Positive and negative regulation of T-cell activation by adaptor proteins.

Adaptor proteins, molecules that mediate intermolecular interactions, are now known to be as crucial for lymphocyte activation as are receptors and effectors. Extensive work from numerous laboratories has identified and characterized many of these adaptors, demonstrating their roles as both positive and negative regulators. Studies into the molecular basis for the actions of these molecules shows that they function in various ways, including: recruitment of positive or negative regulators into signalling networks, modulation of effector function by allosteric regulation of enzymatic activity, and by targeting other proteins for degradation. This review will focus on a number of adaptors that are important for lymphocyte function and emphasize the various ways in which these proteins carry out their essential roles.

Adaptor Proteins, Signal Transducing↗
PubMed1994

The insulin receptor: structure, function, and signaling.

The insulin receptor is a member of the ligand-activated receptor and tyrosine kinase family of transmembrane signaling proteins that collectively are fundamentally important regulators of cell differentiation, growth, and metabolism. The insulin receptor has a number of unique physiological and biochemical properties that distinguish it from other members of this large well-studied receptor family. The main physiological role of the insulin receptor appears to be metabolic regulation, whereas all other receptor tyrosine kinases are engaged in regulating cell growth and/or differentiation. Receptor tyrosine kinases are allosterically regulated by their cognate ligands and function as dimers. In all cases but the insulin receptor (and 2 closely related receptors), these dimers are noncovalent, but insulin receptors are covalently maintained as functional dimers by disulfide bonds. The initial response to the ligand is receptor autophosphorylation for all receptor tyrosine kinases. In most cases, this results in receptor association of effector molecules that have unique recognition domains for phosphotyrosine residues and whose binding to these results in a biological response. For the insulin receptor, this does not occur; rather, it phosphorylates a large substrate protein that, in turn, engages effector molecules. Possible reasons for these differences are discussed in this review. The chemistry of insulin is very well characterized because of possible therapeutic interventions in diabetes using insulin derivatives. This has allowed the synthesis of many insulin derivatives, and we review our recent exploitation of one such derivative to understand the biochemistry of the interaction of this ligand with the receptor and to dissect the complicated steps of ligand-induced insulin receptor autophosphorylation. We note possible future directions in the study of the insulin receptor and its intracellular signaling pathway(s).

Animals↗