Showing papers on "Cooperative binding published in 1990"

Mechanisms of Cooperativity and Allosteric Regulation in Proteins

Abstract: Preface 1. Introduction 2. Haemoglobin: Dependence of allosteric equilibrium on spin state and coordination of the haem iron 3. Haemocyanin: Dependence of allosteric equilibrium on coordination and valency of a binuclear copper complex 4. Haemerythrin: Cooperativity in a binuclear iron complex 5. Glycogen phosphorylase: Control of glycolysis 6. Phosphofructokinase: Further control of glycolysis 7. Feedback inhibition of a biosynthetic pathway: Aspartate Transcarbamoylase 8. Control of nitrogen metabolism: Glutamine synthetase 9. Cooperativity and feedback inhibition without change of quaternary structure: The "trp" and "met" repressors of E. Coli 10. Immunoglobulins: Cooperative binding to multivalent antigens 11. Allosteric membrane proteins.

Role of extracellular disulfide-bonded cysteines in the ligand binding function of the beta 2-adrenergic receptor.

Abstract: Evidence is presented for a role of disulfide bridging in forming the ligand binding site of the {beta}{sub 2}-adrenergic receptor ({beta}AR). The presence of disulfide bonds at the ligand binding site is indicated by competitive inhibition by dithiothreitol (DTT) in radioligand binding assays, by specific protection by {beta}-adrenergic ligands of these effects, and by the requirement of disulfide reduction for limit proteolysis of affinity ligand labeled receptor. The kinetics of binding inhibition by DTT suggest at least two pairs of disulfide-bonded cysteines essential for normal binding. Through site-directed mutagenesis, the authors indeed were able to identify four cysteines which are critical for normal binding affinities and for the proper expression of functional {beta}AR at the cell surface. Unexpectedly, the four cysteines required for normal ligand binding are not those located within the hydrophobic transmembrane domains of the receptor (where ligand binding is presumed to occur) but lie in the extracellular hydrophilic loops connecting these transmembrane segments. These findings indicate that in addition to the well-documented involvement of the membrane-spanning domains of the receptor in ligand binding, there is an important and previously unsuspected role of the hydrophilic extracellular domains in forming the ligand binding site.

Atomic structures of periplasmic binding proteins and the high-affinity active transport systems in bacteria.

Abstract: We have determined and refined the X-ray crystal structures of six periplasmic binding proteins that serve as initial receptors for the osmotic-shock sensitive, active transport of L-arabinose, D-galactose/D-glucose, maltose, sulphate, leucine/isoleucine/valine and leucine. The tertiary structures and atomic interactions between proteins and ligands show common features that are important for understanding the function of the binding proteins. All six structures are ellipsoidal, consisting of two similar, globular domains. The ligand-binding site is located deep in the cleft between the two domains. Irrespective of the nature of the ligand (e.g. saccharide, sulphate dianion or leucine zwitterion), the specificities and affinities of the binding sites are achieved mainly through hydrogen-bonding interactions. Binding of ligands induces a large protein conformational change. Three different structures have been observed among the binding proteins: unliganded 'open cleft', liganded 'open cleft', and liganded 'closed cleft'. Here we discuss the functions of binding proteins in the light of numerous crystallographic and ligand-binding studies and propose a mechanism for the binding protein-dependent, high-affinity active transport.

The adenovirus E4 17-kilodalton protein complexes with the cellular transcription factor E2F, altering its DNA-binding properties and stimulating E1A-independent accumulation of E2 mRNA.

Abstract: E2F is a cellular DNA-binding factor. Its binding activity is changed within adenovirus-infected cells so that it binds cooperatively to pairs of properly spaced and oriented E2F recognition sites. In the work described in this report, the conversion to cooperative binding was shown to require the adenovirus E4 17-kilodalton (kDa) polypeptide. Mutant viruses carrying alterations within the E4 17-kDa coding region failed to generate the infection-specific, cooperatively binding form of E2F. It was possible to alter E2F from uninfected cells so that it bound cooperatively by incubation with a partially purified fraction obtained from infected cells. The E4 17-kDa protein copurified with this activity and was also found to be present in a complex containing E2F. Consistent with its ability to alter the binding of E2F to its recognition sites within the E2 promoter, the E4 17-kDa polypeptide contributed to maximal expression of E2 mRNAs in some cell types. Its ability to enhance E2 transcription did not require expression of the E1A transactivator protein. These results are consistent with a model which proposes that the E4 17-kDa polypeptide binds to the cellular E2F factor, altering its binding behavior and thereby enhancing its ability to stimulate transcription.

Murine interleukin 7 (IL-7) receptor. Characterization on an IL-7-dependent cell line.

Abstract: A murine cell line (IxN/2b) absolutely dependent upon exogenous IL-7 for continued growth has been obtained that expresses lymphoid precursor and class I MHC antigens and also contains a rearranged mu heavy chain. This cell line has been used to define the binding and structural characteristics of the murine IL-7 receptor using 125I-labeled recombinant murine IL-7. 125I-IL-7 binding to IxN/2b cell was rapid and saturable at both 4 degrees and 37 degrees C. Equilibrium binding studies produced curvilinear Scatchard plots at both temperatures with high and low affinity Ka values of approximately 1 x 10(10) M-1 and 4 x 10(8) M-1, respectively, and a total of 2,000-2,500 IL-7 binding sites expressed per cell. Experiments measuring inhibition of binding of 125I-IL-7 by unlabeled IL-7 also produced data consistent with the existence of two classes of IL-7 receptors. Evidence concerning the possible molecular nature of two classes of IL-7 receptors was provided by dissociation kinetics and affinity crosslinking experiments. The dissociation rate of 125I-IL-7 was markedly increased when measured in the presence of unlabeled IL-7 at both 37 degrees and 4 degrees C, which is diagnostic of a receptor population displaying negative cooperativity. Crosslinking studies showed that under both reducing and nonreducing conditions, the major crosslinked species observed corresponded to a receptor size of 75-79 kD while a less intense higher molecular mass crosslinked species was also seen which corresponded to a receptor size approximately twice as large (159-162 kD). Both types of experiments suggest that the IL-7 receptor may form noncovalently associated dimers in the membrane. The IL-7 receptor was expressed on pre-B cells, but not detected on several murine B cell lines or primary mature B cells. It was also expressed on murine thymocytes, some T lineage cell lines, and on bone marrow-derived macrophage. All cells binding 125I-IL-7 exhibited curvilinear Scatchard plots. No cytokines or growth factors tested were able to inhibit binding of 125I-IL-7 to its receptor. These results define the initial binding and structural characteristics, and the cellular distribution, of the murine IL-7 receptor.

Binding of substrate CO2 to the active site of human carbonic anhydrase II: a molecular dynamics study.

Abstract: Molecular dynamics has been used to study binding of substrate CO2 to the active site of human carbonic anhydrase II. Three potential CO2 binding sites have been located. The first is at the active-site hydrophobic pocket (the catalytically productive site), where CO2 is approximately 3.5 A from the zinc ion and interacts with His-94, His-119, Val-121, Val-143, Leu-198, Thr-199, the zinc ion, and the zinc-bound hydroxide ion. The second CO2 binding site is approximately 6 A from the zinc ion, where CO2 interacts with His-64, His-94, Leu-198, Thr-200, Pro-201, Pro-202, and some active-site water molecules. The third CO2 binding site is approximately 10 A from the zinc ion, is largely solvated by water molecules, and interacts with His-64, Asn-67, and Gln-92. At these three CO2 binding sites, the CO2 molecule is highly localized (the average Zn-CO2 distance fluctuation is approximately 1 A) and favors the linear binding orientation toward the zinc ion. This linear binding orientation of CO2 and its electrostatic interaction with the zinc ion direct diffusion of CO2 toward the zinc ion and facilitate the nucleophilic attack from O of the zinc-bound OH- to C of CO2 in the productive hydrophobic binding site. Finally, the two CO2 binding sites outside the hydrophobic binding pocket, which may represent two intermediate states along the CO2 binding pathway, could play important roles as a CO2 relay.

An adenovirus E4 gene product trans-activates E2 transcription and stimulates stable E2F binding through a direct association with E2F.

Abstract: The adenovirus E4 gene encodes a trans-activating function that can stimulate the E2 promoter. E2 promoter sequences required for E4 trans-activation are identical to those required for E1A trans-activation, and these principally are the E2 promoter binding factor (E2F) binding sites. Furthermore, full activation of E2F DNA binding activity requires both E1A and E4 action. Analysis of a series of mutant E4 viruses identifies open reading frame (orf) 6/7 of the E4 transcription unit as that required for activation of E2F binding activity. In addition, the assay of various E4 cDNAs demonstrates that the E4 orf 6/7 also is responsible for the trans-activation of E2 transcription. Translation of the E4 orf 6/7 mRNA, but not a control mRNA, in a reticulocyte extract generates an activity that can stimulate cooperative binding of E2F in vitro, consistent with recent in vivo assays that demonstrate a role for the E4 gene in E2F stable complex formation. This stimulation is due to a direct interaction of the E4 protein with E2F since an antibody that recognizes the E4 orf 6/7 polypeptide detects this E4 protein in the E2F-DNA complex. We conclude that the E4 orf 6/7 product interacts with the E2F factor altering binding to allow formation of a stable complex that results in a stimulation of transcription.

Ca2(+)-dependent binding of tamoxifen to calmodulin isolated from bovine brain.

Abstract: The interaction of the antiestrogen tamoxifen (Tx) with calmodulin (CaM) was investigated by cross-linking between the protein and [ 3 H] tamoxifen aziridine. We observed that CaM binds Tx in a Ca 2+ -dependent manner and that two components are involved in the binding, with apparent dissociation constants ( K d ) of about 6 nm and 9 µm. The high affinity binding site has a maximal capacity of 25 pmol/mg protein, whereas the low affinity binding site has a B max value of 120 nmol/mg protein. The stimulatory effect of Ca 2+ is maximal at the pCa value of 5, and it is noncompetitively inhibited by Mg 2+ . In the micromolar range, the cation-dependent interaction of Tx with CaM exhibits positive cooperativity ( n H = 1.4) and it is specific in the sense that it is inhibited by unlabeled Tx and by the CaM antagonist trifluoperazine. In contrast, no specificity was observed for the Tx binding, which is cation independent. Tx in the nanomolar range forms complexes with CaM which can be visualized by fluorography after electrophoretic separation in a polyacrylamide gel. Furthermore, CaM antagonism of Tx was observed with respect to inhibition of the CaM effect on the RBC membrane (Ca 2+ + Mg 2+ )-ATPase. The results indicate that Tx may alter Ca 2+ -dependent processes by interacting directly with CaM.

Activation of the E2F transcription factor in adenovirus-infected cells involves E1A-dependent stimulation of DNA-binding activity and induction of cooperative binding mediated by an E4 gene product.

Abstract: Previous experiments have demonstrated that the DNA-binding activity of the E2F transcription factor is increased upon adenovirus infection and that both the E1A and E4 genes are required for activation. In this study, we demonstrated that this enhanced binding of E2F to the E2 promoter is the result of two events. (i) There is stimulation of the DNA-binding activity of the E2F factor; this stimulation is E1A dependent but independent of E4. (ii) There is also induction of a stabilized interaction between E2F molecules bound to adjacent promoter sites; induction of stable E2F binding requires E4 gene function. This two-step activation process was also demonstrated in vitro. A heat-stable fraction from extracts of adenovirus-infected cells, which contains the 19-kilodalton E4 protein, was capable of stimulating stable E2F binding in an ATP-independent manner and appeared to involve direct interaction of the E4 protein with E2F. An extract from virus-infected cells devoid of the E4 19-kilodalton protein stimulated E2F DNA binding without forming the stable complex. This reaction required ATP. We conclude that activation of E2F during adenovirus infection is a two-step process involving a change in both the DNA-binding activity of the factor and the capacity to stabilize the interaction through protein-protein contacts.

Structural transitions upon ligand binding in a cooperative dimeric hemoglobin

Abstract: Comparison of the 2.4 angstrom resolution crystal structures of dimeric clam hemoglobin in the deoxygenated and carbon-monoxide liganded states shows how radically different the structural basis for cooperative oxygen binding is from that operative in mammalian hemoglobins. Heme groups are in direct communication across a novel subunit interface formed by the E and F helices. The conformational changes at this interface that accompany ligand binding are more dramatic at a tertiary level but more subtle at a quaternary level than those in mammalian hemoglobins. These findings suggest a cooperative mechanism that links ligation at one subunit with potentiation of affinity at the second subunit.

Cooperativity and specificity in the interactions between DNA and the glucocorticoid receptor DNA binding domain

Abstract: We have employed fluorescence spectroscopy to study the chemical equilibrium between a 115 amino acid protein fragment containing the DNA-binding domain of the human glucocorticoid receptor (DBDr) and a 24-base-pair DNA oligomer containing the glucocorticoid response element (GRE) from the mouse mammary tumor virus promoter region and compared it with the binding to nonspecific DNA at various ionic conditions. We find that binding to both DNAs is cooperative but that DBDr shows a higher affinity for the GRE than for nonspecific DNA and that this difference is more pronounced at increased salt concentrations. Sequence-specific binding to the GRE sequence at 570 mM monovalent cations can be described by a two-site cooperative model, and this supports the notion that DBDr binding to the GRE is enhanced by dimer formation at the recognition site. The product between the (average) association constant for binding to a GRE half-site and the cooperativity parameter was estimated to be K omega = (1-4) x 10(7) M-1 at this salt concentration and 20 degrees C. The sequence-specific binding is not very sensitive to salt concentration in the interval 270-570 mM monovalent cations. However, at lower salt (70 mM) additional binding takes place, presumably nonspecific (cooperative) association to DNA adjacent to the GRE sequence. DBDr binding to nonspecific DNA can be described by the McGhee-von Hippel model for cooperative binding to a chain polymer and is very sensitive to ionic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of the Escherichia coli gal repressor protein with its DNA operators in vitro

Abstract: The binding of Escherichia coli Gal repressor to linear DNA fragments containing two binding sites (OE and OI) within the gal operon was analyzed in vitro with quantitative footprint and mobility-shift techniques. In vivo analysis of the regulation of the gal operon [Haber, R., & Adhya, S. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 9683-9687] has suggested the role of a regulatory "looped complex" mediated by the association of Gal repressor dimers bound at OE and OI. The binding of Gal repressor to a single site can be described by a model in which monomer and dimer are in equilibrium and only the dimer binds to DNA. At pH 7.0, 25 mM KCl, and 20 degrees C, the binding and dimerization free energies are comparable, suggesting that the equilibrium governing the formation of dimers may be important to regulation. The two intrinsic binding constants, delta GI and delta GE, and a constant describing cooperativity, delta GIE, were determined by footprint titration analysis as a function of pH, [KCl], and temperature. Only at 4 and 0 degrees C was delta GIE negative, signifying cooperative binding. These results are thought to be due to a weak dimer to tetramer association interface. delta GE and delta GI had maximal values between pH 6 and pH 7. The dependence of these constants on [KCl] corresponded to the displacement of approximately 2 ion equiv. The temperature dependence could be described by a change in the heat capacity, delta Cp, of -2.3 kcal mol-1 deg-1. Mobility-shift titration experiments conducted at 20 and 0 degrees C yielded values for delta GIE that were consistent with those resolved from the footprint analysis. Unique values of delta GIE were determined by analysis of mobility-shift titrations of Gal repressor with wild-type operator subject to the constraint that delta GE = delta GI: a procedure that eliminates the need to simultaneously analyze wild-type titrations with titrations of OE- and OI- operators.

Analysis of the binding of glyceraldehyde-3-phosphate dehydrogenase to microtubules, the mechanism of bundle formation and the linkage effect

Abstract: The binding of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to microtubules was analysed by the determination of the concentration of the free enzyme in equilibrium with the complex. At low ionic strength (0.03 M) the binding data are best described by a sum of high (Kd = 0.28 microM) and low affinity (Kd = 7.14 microM) sites, showing positive cooperativity. Addition of 1 mM adenosine 5'-[beta,gamma-imido]triphosphate (p[NH]ppA) or increasing the ionic strength to 0.1 M reduces the binding constant of the high-affinity sites considerably. Adding microtubule-associated proteins (at I = 0.1 M) does not appreciably influence the affinities. Total stoichiometries vary over 2.1-1.2 tubulin dimers involved in a binding site for GAPDH. Bundling is reduced concomitantly with the reduction of the affinities and the increase of the stoichiometry to close to 1 mol GAPDH/mol tubulin dimer. The critical concentration of tubulin is practically not influenced by the binding of the enzyme. This behaviour is discussed in terms of the concept of linked functions. p[NH]ppA dissociates the bundles very rapidly. Analytical sedimentation studies showed that the dissociation of the bundles by p[NH]ppA is not due to the dissociation of the tetrameric enzyme. Bundling slows down association and dissociation of microtubules. The rate of bundle formation, after addition of GAPDH to preformed microtubules, is not dependent on the GAPDH concentration.

A novel approach to the generation of high affinity class II-binding peptides.

Abstract: We have found that if core regions crucial for class II binding are incorporated in multiple copies in the same peptide molecule ("reiterative motifs"), marked enhancement of the binding capacity occurs. Isotype specificity (IAd vs IEd binding capacities) is retained in all three antigenic determinants so far analyzed (lambda rep 12-26, OVA 323-339, and hen egg lysozyme 105-120). The mechanism involved in such an effect is not clear, but experiments involving introduction of a peptide spacer between two repeated core regions do not support the notion that the effect is mediated by cross-linking of more than one MHC molecule, favoring the possibility that conformational effects or distinct subsites of interaction on the MHC molecule may be involved. Based on reiterative structures, a peptide molecule composed of only two different amino acids (Ala and His) has been produced that still retains a very high binding affinity. An 125I-radiolabeled form of this peptide has been used to demonstrate that the high binding detected is mediated by the same binding site involved in the interaction of IAd and OVA 323-339. Inhibition of Ag presentation studies further supports the immunologic relevance of the phenomena observed. Finally, we observed naturally occurring clustered binding sites in proximity of immunodominant protein regions, raising the possibility that the phenomenon might have a physiologic counterpart.

Cooperative binding of R17 coat protein to RNA.

Abstract: The binding of the R17 coat protein to synthetic RNAs containing one or two coat protein binding sites was characterized by using nitrocellulose filter and gel-retention assays. RNAs with two available sites bound coat protein in a cooperative manner, resulting in a higher affinity and reduced sensitivity to pH, ionic strength, and temperature when compared with RNAs containing only a single site. The cooperativity can contribute up to -5 kcal/mol to the overall binding affinity with the greatest cooperativity found at low pH, high ionic strength, and high temperatures. Similar solution properties for the encapsidation of the related fr and f2 phage suggest that the cooperativity is due to favorable interactions between the two coat proteins bound to the RNA. This system therefore resembles an intermediate state of phage assembly. No cooperative binding was observed for RNAs containing a single site and a 5' or 3' extension of nonspecific sequence, indicating that R17 coat protein has a very low nonspecific binding affinity. Unexpectedly weak binding was observed for several RNAs due to the presence of alternative conformational states of the RNA.

Apparent lack of MHC restriction in binding of class I HLA molecules to solid-phase peptides.

Abstract: The specificity of binding of solubilized, purified HLA-A,B molecules to solid-phase peptides has been examined using the assay described by Bouillet et al. [1989. Nature (Lond.). 339:473.] 64 peptides derived from the sequences of viral antigens, HLA-A,B,C heavy chains, and clathrin light chains were tested for binding to HLA-A2.1, Aw68.1, Aw69, B44, and B5, molecules that differ by 5-17 residues of the peptide binding groove. 15 of the peptides, including those known to be T cell epitopes, gave significant binding. The pattern of peptide binding for each of the five HLA-A,B molecules was not significantly different. Binding was demonstrated to be a property of native beta 2m-associated HLA-A,B molecules that preserved conformational antigenic determinants after binding to peptide. In comparison to our previous results from solution-based assays the proportion of HLA-A,B molecules that can bind solid-phase peptides is very high. This accessibility of solid-phase peptides to the binding site of MHC molecules may be directly related to the observed absence of MHC specificity in the binding.

Diverse interactions between the individual mutations in a double mutant at the active site of staphylococcal nuclease.

Abstract: In principle, the quantitative effect of a second mutation on a mutant enzyme may be antagonistic, absent, partially additive, additive, or synergistic with respect to the first mutation. Depending on the kinetic or thermodynamic parameter measured, the D21E and R87G mutations of staphylococcal nuclease exhibit four of these five categories of interaction in the double mutant. While Vmax of the R87G single mutant of staphylococcal nuclease is 10(4.8)-fold lower than that of the wild-type enzyme and the Vmax of the D21E single mutant is 10(3.0)-fold below that of wild type, the double mutant D21E + R87G was found to lose a factor of only 10(4.1) in Vmax relative to wild type, rather than the product of the two single mutations (10(7.8)). These results suggest antagonistic structural effects of the individual R87G and D21E mutations. An alternative explanation for the nonadditivity of effects, namely, the separate functioning of these residues in a stepwise mechanism involving the prior attack of water on phosphorus followed by protonation of the leaving group by Arg-87, is unlikely since no enzyme-bound phosphorane intermediate (less than 1% of [enzyme]) was found under steady-state conditions on the R87G mutant by 31P NMR at 242.9 MHz. Like the effects on Vmax, quantitatively similar antagonistic effects of the two mutations were detected on the binding of divalent cations in binary enzyme-Ca2+ and enzyme-Mn2+ complexes and in the ternary enzyme-Ca2(+)-5'-pdTdA complex, suggesting that the effects on Vmax result from antagonistic structural changes at the Ca2+ binding site. Simple additive weakening effects of the two mutations were found on the binding of the substrate 5'-pdTdA, in both the absence and the presence of the divalent cations, Mn2+ and Ca2+. However, synergistic effects of the two mutations were found on the binding of the substrate analogue 3',5'-pdTp, profoundly weakening its binding to the double mutant in both the absence and the presence of divalent cations. Such synergistic effects of the two mutations may result from negative cooperativity or strain in the binding of 3',5'-pdTp to the wild-type enzyme. It is concluded that the quantitative interactions of two active-site mutations of an enzyme can vary greatly depending on which parameter of the enzyme is measured. When the two mutations interact in the same way on several parameters, a common underlying mechanism is suggested.

Nucleotide regulation of Escherichia coli glycerol kinase: initial-velocity and substrate binding studies.

Abstract: Substrate binding to Escherichia coli glycerol kinase (EC 2.7.1.30; ATP-glycerol 3-phosphotransferase) was investigated by using both kinetics and binding methods. Initial-velocity studies in both reaction directions show a sequential kinetic mechanism with apparent substrate activation by ATP and substrate inhibition by ADP. In addition, the Michaelis constants differ greatly from the substrate dissociation constants. Results of product inhibition studies and dead-end inhibition studies using 5'-adenylyl imidodiphosphate show the enzyme has a random kinetic mechanism, which is consistent with the observed formation of binary complexes with all the substrates and the glycerol-independent MgATPase activity of the enzyme. Dissociation constants for substrate binding determined by using ligand protection from inactivation by N-ethylmaleimide agree with those estimated from the initial-velocity studies. Determinations of substrate binding stoichiometry by equilibrium dialysis show half-of-the-sites binding for ATP, ADP, and glycerol. Thus, the regulation by nucleotides does not appear to reflect binding at a separate regulatory site. The random kinetic mechanism obviates the need to postulate such a site to explain the formation of binary complexes with the nucleotides. The observed stoichiometry is consistent with a model for the nucleotide regulatory behavior in which the dimer is the enzyme form present in the assay and its subunits display different substrate binding affinities. Several properties of the enzyme are consistent with negative cooperativity as the basis for the difference in affinities. The possible physiological importance of the regulatory behavior with respect to ATP is considered.

Cooperative homotropic interaction of l‐noradrenaline with the catalytic site of phenylalanine 4‐monooxygenase

Abstract: Catecholamines (adrenaline, noradrenaline and dopamine) are potent inhibitors of phenylalanine 4-monooxygenase (phenylalanine hydroxylase, EC 1.14.16.1). The amines bind to the enzyme by a direct coordination to the high-spin (S = 5/2) Fe(III) at the active site (charge transfer interaction), as seen by resonance Raman and EPR spectroscopy. Experimental evidence is presented that a group with an apparent pKa value of about 5.1 (20 degrees C) is involved in the interaction between the catecholamine and the enzyme. The high-affinity binding of L-noradrenaline to phenylalanine hydroxylase, as studied by equilibrium microdialysis (anaerobically) and ultrafiltration (aerobically), shows positive cooperativity (h = 1.9); at pH 7.2 and 20 degrees C the rat enzyme binds about 0.5 mol L-noradrenaline/mol subunit with a half-maximal binding (S50) at 0.25 microM L-noradrenaline. No binding to the ferrous form of the enzyme was observed. The affinity decreases with decreasing pH, by phosphorylation and by preincubation of the enzyme with the substrate L-phenylalanine, while it increases after alkylation of the enzyme with the activator N-ethylmaleimide. Preincubation of the enzyme with L-phenylalanine also leads to a complete loss of the cooperativity of L-noradrenaline binding (h = 1.0). The many similarities in binding properties of the inhibitor L-noradrenaline and the activator/substrate L-phenylalanine makes it likely that the cooperative interactions of these effectors are due to their binding to the same site. The high-affinity of catecholamines to phenylalanine hydroxylase is a valuable probe to study the active site of this enzyme and is also relevant for the homologous enzyme tyrosine hydroxylase, which is purified as a stable catecholamine-Fe(III) complex.

Metal and anion binding sites in lactoferrin and related proteins

Abstract: The metal and anion binding sites of the protein lactoferrin (Lf) have been defined through crystallographic analyses of FegLf and Cu2Lf. In both cases each metal ion is 6-coordinate, with four protein ligands (2 Tyr, 1 Asp, 1 His) and the synergistic C032- anion which binds as a bidentate ligand. The C032- fits into a pocket between the metal and the N-terminus of an a-helix. Binding of other metals and anions can be understood in terms of the protein structure and a suggested mechanism for binding and release. Differences between the two sites in Lf can also be explained. suggest links between bacterial and mammalian binding proteins. Striking similarities between Lf and a S042- binding protein

Evidence for the importance of a carboxyl group in the binding of ligands to the D2 dopamine receptor.

Abstract: A series of group specific modifying reagents were tested for their effects on [3H]spiperone binding to brain D2 dopamine receptors to identify amino acid residues at the binding site of the D2 dopamine receptor that are critical for ligand binding. The dependence of ligand binding to the receptor on the pH of the incubation medium was also examined. N-Acetylimidazole, 5,5'-dithiobis(2-nitrobenzoic acid), 1,2-cyclohexanedione, and acetic anhydride had no specific effect on [3H]spiperone binding, indicating the lack of participation of tyrosine, free sulphydryl, arginine, or primary amino groups in ligand binding to the receptor. N,N'-Dicyclohexylcarbodiimide (DCCD) potently reduced the number of [3H]spiperone binding sites, indicating that a carboxyl group is involved in ligand binding to the receptor. The effects of DCCD could be prevented by prior incubation of the receptor with D2 dopamine receptor selective compounds. The pH-binding profile for [3H]spiperone binding indicated the importance of an ionising group of pKa 5.2 for ligand binding which may be the same carboxyl group. Diethyl pyrocarbonate, the histidine modifying reagent, also inhibited [3H]spiperone binding, reducing the affinity of the receptor for this ligand but the effects were not at the ligand binding site. From the effects of pH changes on ligand binding some evidence was obtained for a second ionising group (pKa 7.0) that specifically affects the binding of substituted benzamide drugs to the receptor. It is concluded that the D2 dopamine receptor binding site contains separate but over-lapping binding regions for antagonists such as spiperone and substituted benzamide drugs. The former region contains an important carboxyl group; the latter region contains another group that may be a second carboxyl group or a histidine.