Showing papers on "Cooperative binding published in 1993"

Distinct mechanisms for regulation of the interleukin-8 gene involve synergism and cooperativity between C/EBP and NF-kappa B.

Abstract: The interleukin-8 promoter is transcriptionally activated by interleukin-1, tumor necrosis factor alpha, phorbol myristate acetate, or hepatitis B virus X protein through a sequence located between positions -91 and -71. This region contains an NF-kappa B-like and a C/EBP-like binding site. We show here that several members of the NF-kappa B family, including p65, p50, p52, and c-Rel, can bind to this region, confirming an authentic NF-kappa B binding site in the interleukin-8 promoter. Further, C/EBP binds only weakly to the interleukin-8 promoter site. Electrophoretic mobility shift assays with proteins overexpressed in COS cells and with nuclear extracts from tumor necrosis factor alpha-stimulated HeLa cells demonstrated a strong cooperative binding of C/EBP to its site when NF-kappa B is bound to its adjacent binding site. Transfection studies lead to a model that suggests a highly complex regulation of interleukin-8 gene expression at multiple levels: independent binding of C/EBP and NF-kappa B to their respective sites, cooperative binding of C/EBP and NF-kappa B to DNA, and positive synergistic activation through the C/EBP binding site and inhibition through the NF-kappa B binding site by combinations of C/EBP and NF-kappa B. Thus, the ultimate regulation of interleukin-8 gene expression depends on the ratio of cellular C/EBP and NF-kappa B.

L-29, an endogenous lectin, binds to glycoconjugate ligands with positive cooperativity

Abstract: The soluble mammalian lactose-binding lectins L-14-I and L-29 are both secreted and bind to oligosaccharides on laminin, a large extracellular matrix glycoprotein containing polylactosamine chains. Because of the potential functional significance of these lectin-laminin interactions, we compared quantitative aspects of L-14-I and L-29 binding to immobilized laminin using recombinant lectins labeled with 125I. We report that the concentration-dependent binding of L-29 exhibits positive cooperativity whereas binding of L-14-I does not. Cooperative binding of L-29 can also occur on glycoconjugate substrates other than laminin and is not dependent on cystine bond formation or aggregation in solution. L-29 contains repetitive sequences within the N-terminal domain not present in L-14-I. This domain is not required for binding activity, but is required for positive cooperativity. Though the precise mechanism of interaction of L-29 with laminin remains to be determined, it apparently results in assembly of a lectin aggregate on the substrate surface, which could have important functional consequences.

The patterns of binding of RAR, RXR and TR homo- and heterodimers to direct repeats are dictated by the binding specificites of the DNA binding domains.

Abstract: We show here that, in addition to generating an increase in DNA binding efficiency, heterodimerization of retinoid X receptor (RXR) with either retinoic acid receptor (RAR) or thyroid hormone receptor (TR) alters the binding site repertoires of RAR, RXR and TR homodimers. The binding site specificities of both homo- and heterodimers appear to be largely determined by their DNA binding domains (DBDs), and are dictated by (i) homocooperative DNA binding of the RXR DBD, (ii) heterocooperative DNA binding of RXR/RAR and RXR/TR DBDs, and (iii) steric hindrance. No homodimerization domain exists in the DBDs of TR and RAR. The dimerization function which is located in the ligand binding domain further stabilizes, but in general does not change, the repertoire dictated by the corresponding DBD(s). The binding repertoire can be further modified by the actual sequence of the binding site. We also provide evidence supporting the view that the cooperative binding of the RXR/RAR and RXR/TR DBDs to directly repeated elements is anisotropic, with interactions between the dimerization interfaces occurring only with RXR bound to the 5' located motif. This polarity, which appears to be maintained in the full-length receptor heterodimers, may constitute a novel parameter in promoter-specific transactivation.

Binding affinities of synthetic peptides, pyridine-2-carboxamidonetropsin and 1-methylimidazole-2-carboxamidonetropsin, that form 2:1 complexes in the minor groove of double-helical DNA.

Abstract: The designed peptides pyridine-2-carboxamidonetropsin (2-PyN) and 1-methylimidazole-2-carboxamidonetropsin (2-ImN) are crescent-shaped analogs of the natural products netropsin and distamycin A. 2-PyN and 2-ImN bind the 5'-TGTCA-3' sequence as antiparallel side-by-side dimers in the minor groove of DNA. The binding affinities of 2-PyN and 2-ImN to four different 5-bp sites on DNA were determined by quantitative MPE-Fe(II) footprint titration and compared with the tripeptide D from distamycin. The binding affinities of D to the sites 5'-TTTTT-3' and 5'-TGTCA-3' are 2.6 x 10(7) and < 1 x 10(5) M-1, respectively (pH 7.0, 100 mM NaCl). 2-PyN binds these sites with similar affinities, 2.3 x 10(5) and 2.7 x 10(5) M-1, respectively. The affinities of 2-ImN to the same two sites are < 5 x 10(4) and 1.4 x 10(5) M-1, respectively. Substitution of an N-methylpyrrole-2-carboxamide of the distamycin tripeptide by 1-methylimidazole-2-carboxamide has changed the specificities for the two binding sites by a factor of 10(3). The data for 2-PyN and 2-ImN binding the 5'-TGTCA-3' site are best fit by a cooperative binding curve consistent with 2:1 peptide-DNA complexes.

Mutational effects on the cooperativity of Ca2+ binding in calmodulin.

Abstract: The importance of the aspartate ligand in the +Y Ca2+ coordinating position of two EF-hands of calmodulin has been investigated. Synthetic calmodulin genes were used to produce engineered proteins with the wild-type bovine sequence as well as with aspartate 58 in Ca(2+)-binding site II and/or aspartate 95 in site III changed to asparagine. The macroscopic Ca(2+)-binding constants of the intact calmodulins and of tryptic fragments comprising the N- and C-terminal domains were determined from titrations with Ca2+ in the presence of 5,5'-Br2BAPTA. Substitution of aspartate by asparagine in Ca(2+)-binding site II led to a slight increase in the total free energy change on Ca2+ binding, and the cooperativity of Ca2+ binding to the N-terminal sites was substantially increased. The change from aspartate to asparagine in site III decreased the Ca2+ affinity and also appeared to decrease the positive cooperativity between the sites in the C-terminal domain. Thus, identical mutations in sites II and III were found to result in opposite effects. The data imply that involvement of liganding side chains in interactions other than direct calcium attraction and calcium coordination is of considerable importance for the Ca(2+)-binding process, particularly for the cooperativity.

Cooperative binding of the Escherichia coli repressor of biotin biosynthesis to the biotin operator sequence.

Abstract: Regulation of biotin biosynthesis and retention in Escherichia coli depends on a complex set of coupled protein-protein, protein-nucleic acid, and protein-small molecule interactions. The complexity of the biotin system is analogous to that found in gene regulatory systems from other prokaryotes and from eukaryotes. Quantitative understanding of these systems requires thermodynamic studies of the individual contributing interactions. We have initiated such studies of the biotin regulatory interactions. The assembly states of the biotin operon repressor (BirA) and its complex with the allosteric effector, bio-5'-AMP, have been determined by analytical gel filtration chromatography. Both the apo- and holo-repressors are monomeric at protein concentrations several orders of magnitude higher than those required for DNA binding. Results of stoichiometric DNA binding measurements indicate that the BirA-biotin operator (bioO) complex consists of two holo-repressor monomers per operator site. Equilibrium binding of BirA to bioO has been measured using the quantitative DNase footprint technique. Analysis of the data indicates that the binding process is best described by a cooperative model. An upper limit for the cooperative free energy is estimated to be between -2.0 and -3.0 kcal/mol.

Negative cooperativity in the binding of nucleotides to Escherichia coli replicative helicase DnaB protein. Interactions with fluorescent nucleotide analogs

Abstract: The interactions of nucleotides with Escherichia coli replicative helicase DnaB protein have been systematically studied using fluorescent nucleotide analogs, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP), 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP), 3'-O-(N-methylantraniloyl) 5'-diphosphate (MANT-ADP), and 1,N6-ethenoadenosine diphosphate (epsilon ADP). The binding of the analogs is accompanied by strong quenching of the protein fluorescence; 0.76 +/- 0.05, 0.76 +/- 0.05, 0.58 +/- 0.05, and 0.53 +/- 0.5 for TNP-ATP, TNP-ADP, MANT-ADP, and epsilon ADP, respectively. A thermodynamically rigorous method has been applied to obtain all binding parameters from fluorescence titration curves independent of the assumption of strict proportionality between the observed quenching of the protein fluorescence and the degree of nucleotide binding. An exact representation of the observed fluorescence quenching, as a function of the nucleotide binding, is introduced through an empirical function which enables analysis of single binding isotherms without the necessity of determining all quenching constants for different binding sites. Using this method, we determined that, at saturation, the DnaB hexamer binds six molecules of TNP-ATP, TNP-ADP, MANT-ADP, and epsilon ADP, and that there is strong heterogeneity among nucleotide binding sites. The binding isotherms are biphasic. Three molecules of nucleotide are bound in the first high-affinity binding phase, and the subsequent three molecules are bound in the second low-affinity binding phase. The separation of the two binding steps is even more pronounced at higher temperatures. The change of the monitored fluorescence is sequential. The binding of the first nucleotide causes the largest quenching of the protein fluorescence with subsequent nucleotide binding inducing progressively less quenching. The simplest explanation of this behavior is that there is a negative cooperativity among nucleotide binding sites on a DnaB hexamer. The negative cooperativity is an intrinsic property of the DnaB helicase, since it is observed in the binding of nucleotide analogs which are different in type and location of the modifying group. A statistical thermodynamic model is proposed, the hexagon, which provides an excellent description of the binding process using only two interaction parameters, intrinsic binding constant K and cooperativity parameter sigma. The data suggest an important role of the phosphate groups in binding and in recognition of nucleotides by the DnaB helicase.

[3H]resiniferatoxin binding by the vanilloid receptor : species-related differences, effects of temperature and sulfhydryl reagents

Abstract: Specific binding of [3H]resiniferatoxin (RTX) is thought to represent the vanilloid (capsaicin) receptor. In the present study, we have used this binding assay to elucidate the contribution of differential receptor expression to the capsaicin-resistance of hamsters and rabbits; binding parameters were compared to those of species (rats, mice) regarded as capsaicin-sensitive. Whereas the 5-fold lower affinity for [3H]RTX binding in the hamster (100 pM) as compared to the rat (20 pM) is unlikely to account for the 100-fold difference in the in vivo responses of RTX-induced inflammation and hypothermia, the lack of detectable specific [3H]RTX binding sites in the rabbit might represent the predominant mechanism of capsaicin-resistance in this species. Regulation of the vanilloid receptor was further characterized in the rat. In accord with the temperature dependence of both in vivo and in vitro capsaicin actions, we found a marked temperature dependence for association rates. Dissociation turned out to have complex kinetics dependent on time and receptor occupancy. Low pH (5.5-7.0) did not affect receptor binding. Preincubation with heavy metal cations and other sulfhydryl-reactive agents inhibited specific [3H]RTX binding indicating that the vanilloid receptor is a thiol-protein, and that free sulfhydryl groups play an essential role in agonist binding activity. Preliminary characterization suggested noncompetitive inhibition.

Binding of the K+ channel opener [3H]P1075 in rat isolated aorta: relationship to functional effects of openers and blockers.

Abstract: P1075 [N-cyano-N9-(1,1-dimethylpropyl)-N"-3-pyridylguanidine], an analogue of the K+ channel opener pinacidil, was shown to be a K+ channel opener in rat aorta, based on (i) its ability to stimulate 86Rb+ efflux, (ii) its ability to relax contractions in response to noradrenaline under normal conditions (5 mM KCl) but not under depolarized conditions (55 mM KCl), and (iii) the sensitivity of these effects to inhibition by the sulfonylurea glibenclamide. In these assays, P1075 was approximately 20 times more potent than cromakalim. Using a tritiated derivative, [3H] P1075, specific binding could not be detected in microsomal preparations from various tissues. However, in rat aortic strips specific binding of [3H]P1075 has been observed and was reduced by lowering the temperature or by decreasing intracellular ATP levels via metabolic inhibition. Specific [3H]P1075 binding was influenced neither by depolarization (55 mM KCl) nor by lowering the pH from 7.4 to 6.0. Binding was inhibited by representatives from all major families of K+ channel openers, with potencies that correlated well with the potencies obtained in 86Rb+ efflux and relaxation studies. However, stimulation of 86Rb+ efflux occurred at 40 times higher concentrations than did binding (and vasorelaxation). Of the various inhibitors of the K+ channel openers tested, only the sulfonylureas inhibited [3H] P1075 binding with the same rank order of potencies as that required for inhibition of P1075-induced 86Rb+ efflux, although at higher concentrations. The results show that binding of [3H] P1075 is independent of membrane potential but decreases concomitantly with the intracellular ATP level. The excellent correlation between the potencies of the openers and sulfonylurea blockers in binding assays and functional studies suggests that the 9drug receptor9 labeled by [3H]P1075 in rat isolated aorta is of functional relevance. However, the fact that binding of the openers occurred at concentrations considerably lower than those required for K+ channel opening and that binding of the sulfonylureas was only reflected at concentrations higher than those needed to block the channel requires complex models to link binding and effect, possibly involving two agonist binding sites coupled by negative cooperativity.

Tight DNA binding and oligomerization are dispensable for the ability of p53 to transactivate target genes and suppress transformation.

Abstract: The p53 tumor suppressor protein can bind tightly to specific sequence elements in the DNA and induce the transactivation of genes harboring such p53 binding sites. Various lines of evidence suggest that p53 binds to its target site as an oligomer. To test whether oligomerization is essential for the biological and biochemical activities of p53, we deleted a major part of the dimerization domain of mouse wild-type p53. The resultant protein, termed p53wt delta SS, was shown to be incapable of forming detectable homo-oligomers in vitro and is, therefore, likely to be predominantly if not exclusively monomeric. In agreement with the accepted model, p53wt delta SS indeed failed to exhibit measurable DNA binding in vitro. Surprisingly, though, it was still capable of suppressing oncogene-mediated transformation and of transactivating in vivo a target gene containing p53 binding sites. These findings indicate that dimerization-defective p53 is biologically active and may engage in productive sequence-specific DNA interactions in vivo. Furthermore, p53 dimerization probably leads to cooperative binding to specific DNA sequences.

Cooperative binding of an Ultrabithorax homeodomain protein to nearby and distant DNA sites.

Abstract: Cooperativity in binding of regulatory proteins to multiple DNA sites can heighten the sensitivity and specificity of the transcriptional response. We report here the cooperative DNA-binding properties of a developmentally active regulatory protein encoded by the Drosophila homeotic gene Ultrabithorax (Ubx). We show that naturally occurring binding sites for the Ubx-encoded protein contain clusters of multiple individual binding site sequences. Such sites can form complexes containing a dozen or more Ubx-encoded protein molecules, with simultaneous cooperative interactions between adjacent and distant DNA sites. The distant mode of interaction involves a DNA looping mechanism; both modes appear to enhance transcriptional activation in a simple yeast assay system. We found that cooperative binding is dependent on sequences outside the homeodomain, and we have identified regions predicted to form coiled coils carboxy terminal to the homeodomains of the Ubx-encoded protein and several other homeotic proteins. On the basis of our findings, we propose a multisite integrative model of homeotic protein action in which functional regulatory elements can be built from a few high-affinity sites, from many lower-affinity sites, or from sites of some intermediate number and affinity. An important corollary of this model is that even small differences in binding of homeotic proteins to individual sites could be summed to yield large overall differences in binding to multiple sites. This model is consistent with reports that homeodomain protein targets contain multiple individual binding site sequences distributed throughout sizable DNA regions. Also consistent is a recent report that sequences carboxy terminal to the Ubx homeodomain can contribute to segmental specificity.

The primary self-assembly reaction of bacteriophage .lambda. cI repressor dimers is to octamer

Abstract: Cooperative binding of the bacteriophage lambda cI repressor dimer to specific sites of the phage operators OR and OL controls the developmental state of the phage. It has long been believed that cooperativity is mediated by self-assembly of repressor dimers to form tetramers which can then bind simultaneously to adjacent operator sites. As a first step in defining the individual energy contributions to binding cooperativity, sedimentation equilibrium and steady-state fluorescence anisotropy methods have been used to study the higher order assembly reactions of the free repressor in solution. Wild-type repressor with 5-hydroxytryptophan (5-OHTrp) substituted for the native tryptophan [Ross et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12023-12027] and two mutant repressor proteins that bind cooperatively to OR but have altered dimerization properties were also studied. We report here that the primary assembly mode of all four proteins is dimer to octamer. It is not dimer to tetramer as previously assumed. While tetramer does form as an assembly intermediate, dimer-octamer assembly is a concerted process so that tetramer is never a predominant species in solution. Sedimentation velocity experiments suggest that the octamer is highly asymmetric, consistent with an elongated shape. This conformation could allow octamers to bind simultaneously to all three operator sites at either OR or OL. Examination of tetramer and octamer concentrations suggests that both species could be involved in cooperative repressor-operator interactions. Our previous work used the unique spectral properties of 5-OHTrp to demonstrate that octamer binds single-operator DNA and is not dissociated to tetramer [Laue et al. (1993) Biochemistry 32, 2469-2472]. Taken together with the results presented here, octamers as well as tetramers must be considered in developing models to explain the cooperativity of lambda cI repressor binding to operator DNA.

Binding of sodium dodecyl sulfate to a polyelectrolyte based on chitosan

Abstract: The binding behavior of sodium dodecyl sulfate (SDS) to cationic chitosan in free solution is described. Binding isotherm were determined potentiometrically. The results are rationalized by a cooperative binding model for a heterogeneous polyelectrolyte with ionic sequences and nonionic sequences. A theoretical expression for the binding isotherm of a random copolymer is obtained by using Lifson's sequence-generating function (SGF) method. The same binding constant K for chitosan with various degrees of N-acetylation (da) was estimated for the presented model calculation

Isolation of lambda repressor mutants with defects in cooperative operator binding.

Abstract: A hybrid operator-promoter region was designed to aid in a screen for cooperativity mutants of the lambda repressor. In this system, lambda repressor mutants with defects in pairwise cooperative binding are unable to act as efficient transcriptional repressors. Four single amino acid substitutions in the C-terminal domain of the repressor were isolated. Studies of the DNA binding properties of the purified mutant proteins show that a repressor bearing the Gly147-->Asp mutation binds with normal affinity to single operator sites but is defective in pairwise cooperative site binding. Quantitative footprinting studies show that the free energy of interaction between repressor dimers bound at operator sites OR1 and OR2 is reduced from -2.4 kcal/mol for the wild-type repressor to 0 kcal/mol for the GD147 mutant.

Specificity of the binding domain of glucoamylase 1

Abstract: Glucoamylase 1 from Aspergillus niger hydrolyses granular starch at an increased rate due to the presence of a C-terminal starch-binding domain This domain was isolated and shown to bind to the malto-oligosaccharides Glc2 to Glc11 with a stoichiometry of 1 mol ligand/mol protein The affinity for these ligands increased with increasing degree of polymerisation until Glc9, above which no further increase was observed We suggest that this indicates that for maximum affinity the substrate should be able to form a helical conformation, which mimics the conformation of amylose in granular starch We propose a model of how the complex between the malto-oligosaccharides and the binding domain is formed and indicate how this affects the differences in binding modes for soluble and insoluble substrates Glucono-1,5-lactone interacts with the binding domain at a different site to the malto-oligosaccharides allowing the formation of a ternary complex between the binding domain, a malto-oligosaccharide and glucono-1,5-lactone The binding domain also binds to linear α-1,6-linked glucose digosaccharides (dextran), but with much lower affinity than for α-1,4-linked glucose This ligand appears to interact with the binding domain at both binding sites, i e at the site to which the malto-oligosaccharides bind and also at the site to which glucono-1,5-lactone binds The relevance of the results to the mechanism of action of other polysaccharide-hydrolysing enzymes containing both a catalytic and a binding domain is discussed

Multiple binding modes of the single-stranded DNA binding protein from Escherichia coli as detected by tryptophan fluorescence and site-directed mutagenesis

Abstract: We have systematically substituted the four tryptophan residues of the single-stranded DNA binding protein from Escherichia coli (EcoSSB) by polar (serine or threonine) and aromatic (tyrosine or phenylalanine) amino acids. The resulting mutants with either single amino acid exchanges or triple substitutions are all active in ssDNA binding, though in some cases with reduced affinities. Measurements of the fluorescence of the mutated EcoSSBs show that there is no interaction between the four different tryptophan residues. We analyzed the ssDNA binding of the mutant proteins by fluorescence titrations. At 0.3 M NaCl ("high salt"), all singly substituted proteins bind to poly(dT) in a manner comparable to wild-type EcoSSB, covering 65 nucleotides with 1 EcoSSB tetramer. W54S mutant protein is an exception since even at 0.3 M NaCl it covers approximately 35 nucleotides, a behavior which is typical of salt concentrations below 10 mM NaCl ("low salt"). From this observation, it is inferred that tryptophan-54 is involved in a direct interaction with the ssDNA favoring the "high-salt" binding mode. All mutant proteins lacking tryptophan-54 but possessing tryptophan-88 at "low-salt" concentrations show a nonmonotonous behavior in the fluorescence titrations. This behavior can be interpreted assuming a model of cooperative binding of EcoSSB to poly(dT) with two different binding site sizes (n approximately 27 and n approximately 33) and different binding affinities. A quantitative treatment of the problem of multiple binding modes in the interaction of a multidentate ligand with a linear polymer is applied to these titrations.

Ligand-mediated modulation of estrogen receptor conformation by estradiol analogs

Abstract: The studies presented here show how changing the structure of the ligand can affect the conformation of the receptor. Five different estradiol analogs have been tested for binding to the calf uterine estrogen receptor. In three of the analogs the phenolic hydroxyl group had been moved from the 3 to the 1, 2, or 4 position on the A-ring (1-hydroxyestratrien-17 beta-ol, 2-hydroxyestratrien-17 beta-ol, or 4-hydroxyestratrien-17 beta-ol). In the remaining two analogs either the A- or the D-ring hydroxyl group had been removed altogether (estratrien-17 beta-ol or 3-hydroxyestratriene). Competition binding assay showed that the relative binding affinity for the estrogen receptor had been weakened by all changes in the structure of the ligand. Furthermore, the ligands in which either the 3 beta- or the 17 beta-hydroxyl group was missing produced nonparallel slopes in the linear portions of the displacement curves compared to that of estradiol; the ligands in which the phenolic hydroxyl had simply been moved around the A-ring, however, did not. These observations implied that the receptor binding mechanism used by the monohydroxyl ligands was different from that of estradiol. Saturation binding analysis showed that while the presence of any of the dihydroxyl ligands or that of estratrien-17 beta-ol decreased the positive cooperativity of the [3H]estradiol-estrogen receptor interaction, the presence of the 3-hydroxyestratriene ligand increased it. These results suggest that both the binding mechanism and the affinity of the ligand for the receptor are exquisitely sensitive to the structure of the ligand.

Vanilloid (capsaicin) receptor in the rat: positive cooperativity of resiniferatoxin binding and its modulation by reduction and oxidation.

Abstract: Specific [3H]resiniferatoxin (RTX) binding is thought to represent the postulated vanilloid (capsaicin) receptor. In the present report, this binding has been reevaluated using a modified [3H] RTX binding assay in which the high nonspecific binding, which limited the previous characterization, was reduced by adding alpha 1-acid glycoprotein, a plasma protein that binds RTX, to the usual binding assay after RTX binding by the vanilloid receptor had been terminated. Specific [3H]RTX binding by both dorsal root ganglion (DRG) and spinal cord membranes of the rat followed sigmoidal saturation kinetics indicating apparent positive cooperativity. The cooperativity index determined by fitting the data to the Hill equation was 1.7 in DRG and 1.9 in spinal cord. Apparent dissociation constants were estimated as 24 pM for DRG and 11 pM for spinal cord preparations. As predicted by the modified Hill equation, at low receptor occupancy nonradioactive agonists (RTX, tinyatoxin, capsaicin) produced biphasic competition curves. The initial (enhancement) phase of these curves correlated with the biological potency of the agonist. Dithiothreitol reduced both positive cooperativity and apparent binding affinity; the oxidizing agent 5,5'-dithiobis-(2-nitrobenzoic acid) reduced the cooperativity index without a major effect on binding affinity. These findings suggest that the vanilloid receptor is a receptor cluster in which the subunits cooperate; cooperation is, at least in part, subject to redox modulation.

Mutation of lysine 233 to alanine introduces positive cooperativity into tyrosyl-tRNA synthetase.

Abstract: Tyrosyl-tRNA synthetase from Bacillus stearothermophilus is a dimeric enzyme which displays half-of-sites reactivity with respect to the binding of both tyrosine and ATP. The binding of both substrates follows Michaelis-Menten kinetics. Mutation of lysine 233 to alanine (K233A) decreases the affinity of the active subunit for ATP at both saturating and subsaturating tyrosine concentrations (from the Hill plot, kcat = 0.56 s-1, nH = 1.54, Kd = 372 mM at 50 microM tyrosine). In addition, this mutant displays sigmoidal kinetics (characteristic of positive cooperativity) with respect to the binding of ATP. These two effects can be reversed by the addition of NaCl (0.5 m final concentration) or by a second alanine mutation at either position K230 or T234. The effect of either NaCl or second site mutation is to increase the binding affinity of the K233A mutant for ATP (KATP values are 22 mM for the K233A mutant in the presence of 0.5 M NaCl, 0.16 mM for the K230A/K233A mutant, and 0.14 mM for the K233A/T234A mutant). With the restoration of the tight binding of ATP, Michaelis-Menten kinetics are restored since the kinetic analysis of tyrosyl adenylate formation involves only binding of ATP to the active subunit. It is likely that the physical mechanism for the positive cooperativity present in the K233A mutant actually exists in the wild-type enzyme but is not observed kinetically due to the initial binding of ATP to the active subunit. These results indicate that, in some cases, a decrease in substrate affinity is sufficient to introduce cooperativity into a noncooperative enzyme.