Site-directed mutagenesis of the greasy slide aromatic residues within the LamB (maltoporin) channel of Escherichia coli: effect on ion and maltopentaose transport

The 3D-structure of the maltooligosaccharide-specific LamB-channel of Escherichia coli (also called maltoporin) is known from X-ray crystallography. The 3D structure suggests that a number of aromatic residues (Y6, Y41, W74, F229, W358 and W420) within the channel lumen are involved in carbohydrate and ion transport. All aromatic residues were replaced by alanine-scanning mutagenesis. Furthermore, LamB mutants were created in which two, three, four, five and all six aromatic residues were replaced to study their effects on ion and maltopentaose transport through LamB. The purified mutant proteins were reconstituted into lipid bilayer membranes and the single-channel conductance of the mutants was studied in conductance experiments. The results suggest that all aromatic residues provide some steric hindrance for ion transport through LamB. Highest impact is provided by Y6 and Y41 that are localized opposite Y118, which form the central constriction of the LamB channel. Stability constants for binding of maltopentaose to the mutant channels were measured using titration experiments with the carbohydrate. The mutation of one or several aromatic residue(s) led to a substantial decrease of the stability constant of binding. The highest effect was observed when all aromatic residues were replaced by alanine because no binding of maltopentaose could be detected in such a case. However, binding was again possible when Y118 was replaced by tryptophan. The carbohydrate-induced block of the channel function could be used also for the study of current noise through the different mutant LamB-channels. The analysis of the power density spectra of some of the mutants allowed the evaluation of the on-rate and off-rate constants (k1 and k(-1)) of carbohydrate binding to the binding site inside the channels. The results suggest that both on-rate and off-rate constants were affected by the mutations. For most mutants, k1 decreased and k(-1) increased. The possible influence of the aromatic residues of the greasy slide on carbohydrate and ion transport through LamB is discussed.     

A quantitative study of the interactions of Bacillus anthracis edema factor and lethal factor with activated protective antigen

Bacillus anthracis secretes three proteins, which associate in binary combinations to form toxic complexes at the surface of mammalian cells. Receptor-bound protective antigen (PA) is proteolytically activated, yielding a 63 kDa fragment (PA(63)). PA(63) oligomerizes into heptamers, which bind edema factor (EF) or lethal factor (LF) to form the toxic complexes. We undertook a quantitative analysis of the interactions of EF with PA(63) by means of surface plasmon resonance (SPR) measurements. Heptameric PA(63) was covalently bound by amine coupling to an SPR chip, or noncovalently bound via a C-terminal hexahistidine tag on the protein to Ni(2+)nitrilotriacetate groups on the chip. Values of k(on) and k(off) for EF at 23 degrees C were approximately 3 x 10(5) M(-)(1) s(-)(1) and (3-5) x 10(-)(4) s(-)(1), respectively, giving a calculated K(d) of (1-2) x 10(-)(9) M. A similar value of K(d) (7 x 10(-)(10) M) was obtained when we measured the binding of radiolabeled EF to receptor-bound PA(63) on the surface of L6 cells (at 4 degrees C). Each of these analyses was also performed with LF and LF(N) (the N-terminal 255 residues of LF), and values obtained were comparable to those for EF. The similarity in the dissociation constants determined by SPR and by measurements on the cell surface suggests that the presence of the receptor does not play a large role in the interaction between PA(63) and EF/LF.     

Mechanism of C2-toxin inhibition by fluphenazine and related compounds: investigation of their binding kinetics to the C2II-channel using the current noise analysis

The binding component C2II of the binary actin ADP-ribosylating C2-toxin from Clostridium botulinum is essential for intoxication of target cells. Activation by a protease leads to channel formation and this is presumably required for the transport of the toxic C2I component into cells. The C2II-channel is cation selective and contains a binding site for fluphenazine and structurally related compounds. Ion transport through C2II and in vivo intoxication is blocked when the sites are occupied by the ligands. C2II was reconstituted into artificial lipid bilayer membranes and formed ion permeable channels. The binding constant of chloroquine, primaquine, quinacrine, chloropromazine and fluphenazine to the C2II-channel was determined using titration experiments, which resulted in its block. The ligand-induced current noise of the C2II-channels was investigated using fast Fourier transformation. The noise of the open channels had a rather small spectral density, which was a function of the inverse frequency up to about 100 Hz. Upon addition of ligands to the aqueous phase the current through C2II decreased in a dose-dependent manner. Simultaneously, the spectral density of the current noise increased drastically and its frequency dependence was of Lorentzian type, which was caused by the on and off-reactions of the ligand-mediated channel block. The ligand-induced current noise of C2II was used for the evaluation of the binding kinetics for different ligands to the channel. The on-rate constant of ligand binding was between 10(7) and 10(9) M(-1) s(-1) and was dependent on the ionic strength of the aqueous phase. The off-rate varied between about 10 s(-1) and 3900 s(-1) and depended on the structure of the ligand. The role of structural requirements for the effective block of C2II by the different ligands is discussed.     

Interaction of the 20 kDa and 63 kDa fragments of anthrax protective antigen: kinetics and thermodynamics

The action of anthrax toxin begins when the protective antigen (PA(83), 83 kDa) moiety binds to a mammalian cell-surface receptor and is cleaved by a furin-family protease into two fragments: PA(20) (20 kDa) and PA(63) (63 kDa). After PA(20) dissociates, receptor-bound PA(63) spontaneously oligomerizes to form a heptameric species, which is able to bind the two enzymatic components of the toxin and transport them to the cytosol. Treatment of PA(83) with trypsin yielded PA(63) and a form of PA(20) lacking unstructured regions at the N- and C-termini. We labeled these fragments with dyes capable of fluorescence resonance energy transfer to quantify their association in solution. We kinetically determined that the equilibrium dissociation constant is 190 nM with a dissociation rate constant, k(off), of 3.3 x 10(-)(2) s(-)(1) (t(1/2) of 21 s). A two-step association process was observed using stopped-flow: a fast bimolecular step (k(on) = 1.4 x 10(5) M(-)(1) s(-)(1)) was followed by a slower unimolecular step (k = 3.5 x 10(-)(3) s(-)(1)) with an equilibrium isomerization constant, K(iso), of 2.1. The two-step mechanism most consistent with the data is one in which the dissociation of the PA(20).PA(63) complex is followed by an isomerization in the PA(63) moiety. Our results indicate that, following the cleavage of PA on the cell surface, PA(20) is largely dissociated within a minute. A slow isomerization step in PA(63) may then potentiate it for oligomerization and subsequent steps in toxin action.     

Anthrax lethal factor (LF) mediated block of the anthrax protective antigen (PA) ion channel: effect of ionic strength and voltage

The anthrax toxin complex consists of three different molecules, protective antigen (PA), lethal factor (LF), and edema factor (EF). The activated form of PA, PA(63), forms heptamers that insert at low pH in biological membranes forming ion channels and that are necessary to translocate EF and LF in the cell cytosol. LF and EF are intracellular active enzymes that inhibit the host immune system promoting bacterial outgrowth. Here, PA(63) was reconstituted into artificial lipid bilayer membranes and formed ion-permeable channels. The heptameric PA(63) channel contains a binding site for LF on the cis side of the channel. Full-size LF was found to block the PA(63) channel in a dose- and ionic-strength-dependent way with half-saturation constants in the nanomolar concentration range. The binding curves suggest a 1:1 relationship between (PA(63))(7) and bound LF that blocks the channel. The presence of a His(6) tag at the N-terminal end of LF strongly increases the affinity of LF toward the PA(63) channel, indicating that the interaction between LF and the PA(63) channel occurs at the N terminus of the enzyme. The LF-mediated block of the PA(63)-induced membrane conductance is highly asymmetric with respect to the sign of the applied transmembrane potential. The result suggested that the PA(63) heptamers contain a high-affinity binding site for LF inside domain 1 or the channel vestibule and that the binding is ionic-strength-dependent.     

Characterization of the vascular endothelial growth factor-receptor interaction and determination of the recombinant protein by an optical receptor sensor

Vascular endothelial growth factor (VEGF) is one of the most important factors controlling angiogenesis. It is a homodimeric glycoprotein belonging to the family of cysteine-knot proteins. The biological activity is transduced via membrane-spanning receptors of the tyrosine kinase receptor family. Each biologically active VEGF has two receptor binding sites leading to receptor dimerization as first step following ligand binding. The ligand-binding site of the receptor is localized on extracellular Ig-like domains. The extracellular part of the receptor Flt-1 (VEGFR-1) was expressed as soluble protein and was used as receptor in an optical affinity sensor system (BIAcore). Suitable conditions allowed the determination of the association and dissociation rate constants as k(a)=4+/-1.2 x 10(6) M(-1) s(-1) and k(d)=3+/-0.8 x 10(-5) s(-1), respectively, leading to an affinity constant of K(D)=7.5+/-3 pM, which is within the range published already from other investigations and methods. Increasing receptor loadings of the sensor surface decreased the binding efficiency, as the ratio of bound VEGF-molecules to theoretically available binding sites increased from 1:1.5 to 1:2.6. Increasing the surface loading further, allowed the establishment of a quantitative assay with the analytical performance being influenced by the receptor loading and the contact time between sample and immobilized receptor, i.e. sample volume. This assay was used for VEGF determination during the cultivation of a recombinant Pichia pastoris strain.     

Tight binding of inhibitors to bovine bc1 complex is independent of the Rieske protein redox state. Consequences for semiquinone stabilization in the quinol oxidation site

To determine the effect of the redox state of the Rieske protein on ligand binding to the quinol oxidation site of the bc(1) complex, we measured the binding rate constants (k(1)) for stigmatellin and myxothiazol, at different concentrations of decylbenzoquinone or decylbenzoquinol, in the bovine bc(1) complex with the Rieske protein in the oxidized or reduced state. Stigmatellin and myxothiazol bound tightly and competitively with respect to quinone or quinol, independently of the redox state of the Rieske protein. In the oxidized bc(1) complex, the k(1) values for stigmatellin ( approximately 2.6 x 10(6) m(-1)s(-1)) and myxothiazol ( approximately 8 x 10(5) m(-1)s(-1)), and the dissociation constant (K(d)) for quinone, were similar between pH 6.5 and 9, indicating that ligand binding is independent of the protonation state of histidine 161 of the Rieske protein (pK(a) approximately 7.6). Reduction of the Rieske protein increased the k(1) value for stigmatellin and decreased the K(d) value for quinone by 50%, without modifying the k(1) for myxothiazol. These results indicate that reduction of the Rieske protein and protonation of histidine 161 do not induce a strong stabilization of ligand binding to the quinol oxidation site, as assumed in models that propose the existence of a highly stabilized semiquinone as a reaction intermediate during quinol oxidation.     

Palmitate binding to and efflux kinetics from human erythrocyte ghost

At 0 degrees C, pH 7.3, palmitate (PA) binds to human erythrocyte ghosts suspended in 0.2% bovine serum albumin (BSA) solution with molar ratios of PA to BSA, v, between 0.2 and 1.3. The binding depends on the water phase PA concentration, measured in equilibrium experiments, using BSA-filled ghosts as semipermeable bags. The saturable binding has a capacity of 19.4 +/- 7.5 nmol g-1 packed ghosts (7.2 x 10(9) cells) and Kd = 13.5 +/- 5 nM. PA exchange efflux kinetics to 0.2% BSA is recorded from ghosts without and with 0.2% BSA with a resolution time of about 1 s. Data are analyzed in terms of compartmental models. Using BSA-free ghosts the kinetics is essentially monoexponential. The rate constant is 0.0287 +/- 0.0022 s-1. Using ghosts with BSA, the kinetics is biexponential with widely different rate constants. Extrapolated zero-time values reflect, according to additional investigations, 'instantaneous' release of PA from the outer surface of the ghosts. Analyses of the biexponential curve up to about 55% tracer efflux assign unequivocally values to three model parameters. (1) k1, the dissociation rate constant of the PA-BSA complex is (1.47 +/- 0.03) x 10(-3) s-1 and (2.56 +/- 0.08) x 10(-3) s-1 and (4.08 +/- 0.13) x 10(-3) s-1 at v = 0.2, 0.6 and 1.4, respectively. (2) k3*, the overall rate constant of PA transport from the inside of the ghost membrane to the medium is 0.0269 +/- 0.0020 s-1 independent of v. (3) Qkin, the ratio of PA on the inside of the membrane to PA on BSA within the ghosts is v dependent and smaller than a corresponding ratio Qeq measured in equilibrium by a value corresponding to PA on the outer surface. This fraction is released with a rate constant, k5, which is of the order of 1 s-1. The data suggest a maximum PA transport capacity, Jmax, of 2 pmol min-1 cm-2, 0 degrees C, pH 7.3.     

Site-directed mutagenesis of tyrosine 118 within the central constriction site of the LamB (maltoporin) channel of Escherichia coli. II. Effect on maltose and maltooligosaccharide binding kinetics

The 3-D structure of the maltooligosaccharide-specific LamB channel of Escherichia coli (also called maltoporin) is known from x-ray crystallography. The central constriction of the channel formed by the external loop 3 is controlled by tyrosine 118. Y118 was replaced by site-directed mutagenesis by 10 other amino acids (alanine (A), isoleucine (I), asparagine (N), serine (S), cysteine (C), aspartic acid (D), arginine (R), histidine (H), phenylalanine (F), and tryptophan (W)) including neutral ones, negatively and positively charged amino acids to study the effect of their size, their hydrophobicity index, and their charge on maltose and maltooligosaccharide binding to LamB. The mutants were reconstituted into lipid bilayer membranes and the stability constants for binding of maltose, maltotriose, maltopentaose, and maltoheptaose to the channel were measured using titration experiments. The mutation of Y118 to any other non-aromatic amino acid led to a substantial decrease of the stability constant of binding by factors between about two and six. The highest effect was observed for the mutant Y118A. Replacement of Y118 by the two other aromatic amino acids, phenylalanine (F) and tryptophan (W), resulted in a substantial increase of the stability constant maximally by a factor of almost 400 for the Y118W mutant. The carbohydrate-induced block of the channel function was used for the study of current noise through the different mutant LamB channels. The analysis of the power density spectra allowed the evaluation of the on- and off-rate constants (k(1) and k(-1)) of sugar binding. The results suggest that both rate constants were affected by the mutations. For most mutants, with the exception of Y118F and Y118W, k(1) decreased and k(-1) increased, whereas the opposite was found for the aromatic amino acid mutants. The results suggest that tyrosine 118 has a crucial effect on carbohydrate transport through LamB.     

CymA of Klebsiella oxytoca outer membrane: binding of cyclodextrins and study of the current noise of the open channel

CymA, the outer membrane component of the cyclodextrin (CD) uptake and metabolism system of Klebsiella oxytoca, was reconstituted into lipid bilayer membranes. The channel properties of this unusual porin were studied in detail. The binding of CDs to the channel resulted in its complete block for ion transport. This result allowed the detailed investigation of carbohydrate binding, and the stability constants for the binding of cyclic and linear carbohydrates to the binding site inside the channel were calculated from titration experiments of the membrane conductance with the carbohydrates. Highest stability constant was observed for alpha-cyclodextrin (alpha-CD; K = 32,000 1/M) followed by beta-cyclodextrin (beta-CD; K = 1970 1/M) and gamma-cyclodextrin (gamma-CD; K = 310 1/M). Linear maltooligosaccharides bound also to CymA but with much smaller stability constants as compared to cyclic ones. The noise of the current through CymA in multi- and single-channel experiments was investigated using fast Fourier transformation. The current through the open channels had a rather high spectral density, which was a Lorentzian function of the frequency up to 2000 Hz. Upon addition of cyclic dextrins to the aqueous phase the spectral density decreased in a dose-dependent manner, which made it impossible to evaluate the binding kinetics. Experiments with single CymA-channels demonstrated the channel is highly asymmetric concerning channel flickers and current noise.     

The observation of multivalent complexes of Shiga-like toxin with globotriaoside and the determination of their stoichiometry by nanoelectrospray Fourier-transform ion cyclotron resonance mass spectrometry.

We show by nanoelectrospray ionization (nanoES) Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) that it is possible to observe oligosaccharide-protein complexes with dissociation constants in the millimolar range, such as P(k) trisaccharide (globotriaoside) complexed with the Shiga-like toxin (SLT) of pathogenic E. coli. It is further demonstrated that nanoES/FT-ICR MS is an exquisite method to study quantitative aspects of the association of mono- and polyvalent oligosaccharide ligands with multimeric proteins, such as the SLTs. At increasing trisaccharide:protein ratios it was shown that the B(5 )toxin subunit complexes with 5 P(k) trisaccharides and only after all 5 copies of site 2 are essentially filled do any of the remaining 10 receptor sites become occupied. From the distribution of bound P(k)'s at the five binding sites, it was possible to establish association constants for each of the five sites and to confirm that binding occurs noncooperatively, the association constants for each site are identical and that compared to site 1, site 2 exhibits a tenfold higher affinity for the globotriaoside synthetic ligand 1. The facile identification of the occupancy of binding sites represents information that is not readily available by other techniques. This sensitive and rapid estimation of association constants for protein-ligand complexes, which are free of unpredictable secondary effects that plague enzyme linked assays, is likely to find wide application.     

Increased stability and lifetime of the complex formed between DNA and meta-phenyl-substituted Hoechst dyes as studied by fluorescence titrations and stopped-flow kinetics

The large increase in fluorescence upon binding of five para- and meta-phenyl substituted hydroxy and methoxy derivatives of the Hoechst dye with poly[d(A-T)], d(CGCGAATTCGCG)2, and its corresponding T4-looped 28-mer hairpin was used to monitor the binding by equilibrium titrations and by stopped-flow kinetics. The affinity increases in the same order for the three DNAs: p-OH相似文献   

Binding of N-terminal fragments of anthrax edema factor (EF(N)) and lethal factor (LF(N)) to the protective antigen pore

Anthrax toxin consists of three different molecules: the binding component protective antigen (PA, 83 kDa), and the enzymatic components lethal factor (LF, 90 kDa) and edema factor (EF, 89 kDa). The 63 kDa C-terminal part of PA, PA(63), forms heptameric channels that insert in endosomal membranes at low pH, necessary to translocate EF and LF into the cytosol of target cells. In many studies, about 30 kDa N-terminal fragments of the enzymatic components EF (254 amino acids) and LF (268 amino acids) were used to study their interaction with PA(63)-channels. Here, in experiments with artificial lipid bilayer membranes, EF(N) and LF(N) show block of PA(63)-channels in a dose, voltage and ionic strength dependent way with high affinity. However, when compared to their full-length counterparts EF and LF, they exhibit considerably lower binding affinity. Decreasing ionic strength and, in the case of EF(N), increasing transmembrane voltage at the cis side of the membranes, resulted in a strong decrease of half saturation constants. Our results demonstrate similarities but also remarkable differences between the binding kinetics of both truncated and full-length effectors to the PA(63)-channel.     

Investigation of the alpha(1)-glycine receptor channel-opening kinetics in the submillisecond time domain.

The activation and desensitization kinetics of the human alpha(1)-homooligomeric glycine receptor, which was transiently expressed in HEK 293 cells, were studied with a 100-microseconds time resolution to determine the rate and equilibrium constants of individual receptor reaction steps. Concentration jumps of the activating ligands glycine and beta-alanine were initiated by photolysis of caged, inactive precursors and were followed by neurotransmitter binding, receptor-channel opening, and receptor desensitization steps that were separated along the time axis. Analysis of the ligand concentration-dependence of these processes allows the determination of 1) the rate constants of glycine binding, k(+1) approximately 10(7) M(-1) s(-1), and dissociation, k(-1) = 1900 s(-1); 2) the rates of receptor-channel opening, k(op) = 2200 s(-1), and closing, k(cl) = 38 s(-1); 3) the receptor desensitization rate, alpha = 0.45 s(-1); 4) the number of occupied ligand binding sites necessary for receptor-channel activation and desensitization, n >/= 3; and 5) the maximum receptor-channel open probability, p(0) > 0.95. The kinetics of receptor-channel activation are insensitive to the transmembrane potential. A general model for glycine receptor activation explaining the experimental data consists of a sequential mechanism based on rapid ligand-binding steps preceding a rate-limiting receptor-channel opening reaction and slow receptor desensitization.     

Noise analysis of ion current through the open and the sugar-induced closed state of the LamB channel of Escherichia coli outer membrane: evaluation of the sugar binding kinetics to the channel interior.

LamB, a sugar-specific channel of Escherichia coli outer membrane was reconstituted into lipid bilayer membranes and the current noise was investigated using fast Fourier transformation. The current noise through the open channels had a rather small spectral density, which was a function of the inverse frequency up to about 100 Hz. The spectral density of the noise of the open LamB channels was a quadratic function of the applied voltage. Its magnitude was not correlated to the number of channels in the lipid bilayer membrane. Upon addition of sugars to the aqueous phase the current decreased in a dose-dependent manner. Simultaneously, the spectral density of the current noise increased drastically, which indicated interaction of the sugars with the binding site inside the channel. The frequency dependence of the spectral density was of Lorentzian type, although the power of its frequency dependence was not identical to -2. Analysis of the power density spectra using a previously proposed simple model (Benz, R., A. Schmid, and G. H. Vos-Scheperkeuter. 1987. J. Membr. Biol. 100: 12-29), allowed the evaluation of the on- and the off-rate constants for the maltopentaose binding to the binding site inside the LamB channels. This means also that the maltopentaose flux through the LamB channel could be estimated by assuming a simple one-site, two-barrier model for the sugar transport from the results of the noise analysis.     

单链尿激酶型纤溶酶原激活剂Kringle结构域催化功能探讨

 Kringle(K)结构域广泛存在于与凝血和纤溶相关的各种因子中 .虽然 K结构域在一级结构上是一种比较保守的结构域 (与其他结构域相比 ) ,但是 K结构域的功能在各种因子中的作用有很大的区别 .为探讨单链尿激酶型纤溶酶原激活剂 (scu- PA)的 K结构域功能 ,构建了在 scu- PA的 K结构域的 1 1 8位 Gly与 1 1 9位 Leu之间插入 PRGDWR序列的突变体 (称为 insert mutant B,In B) ,并测定了野生型 scu- PA与 In B的纤溶相关反应的动力学常数 . scu- PA与 In B水解 S- 2 4 4 4反应的 Km 值无明显变化 (分别为 60 .4与 56.8μmol·L-1) ,而 In B的 kcat值 (0 .33s-1)比 scu- PA的 kcat值 (7.31 s-1)降低很多 ;In B激活纤溶酶原反应的 Km 值 (0 .397μmol· L-1)比 scu- PA Km 值(0 .648μmol·L-1)降低 40 % ,但 kcat值 (0 .0 1 65s-1)比 scu- PA的 kcat值 (0 .0 62 6s-1)降低 74% .以上结果说明 :K结构域主要与反应活性相关 ,而与酶及底物的亲和性无关 .     

Kinetic characterization of catalysis by the chemotaxis phosphatase CheZ. Modulation of activity by the phosphorylated CheY substrate

CheZ catalyzes the dephosphorylation of the response regulator CheY in the two-component regulatory system that mediates chemotaxis in Escherichia coli. CheZ is a homodimer with two active sites for dephosphorylation. To gain insight into cellular mechanisms for the precise regulation of intracellular phosphorylated CheY (CheYp) levels, we evaluated the kinetic properties of CheZ. The steady state rate of CheZ-mediated dephosphorylation of CheYp displayed marked sigmoidicity with respect to CheYp concentration and a k(cat) of 4.9 s(-1). In contrast, the gain of function mutant CheZ-I21T with an amino acid substitution far from the active site gave hyperbolic kinetics and required far lower CheYp for half-saturation but had a similar k(cat) value as the wild type enzyme. Stopped flow fluorescence measurements demonstrated a 6-fold faster CheZ/CheYp association rate for CheZ-I21T (k(assoc) = 3.4 x 10(7) M (-1) s(-1)) relative to wild type CheZ (k(assoc) = 5.6 x 10(6) M(-1) s(-1)). Dissociation of the CheZ.CheYBeF(3) complex was slow for both wild type CheZ (k(dissoc) = 0.040 s(-1)) and CheZ-I21T (k(dissoc) = 0.023 s(-1)) and, when taken with the k(assoc) values, implied K(d) values of 7.1 and 0.68 nm, respectively. However, comparison of the k(dissoc) and k(cat) values implied that CheZ and CheYp are not at binding equilibrium during catalysis and that once CheYp binds, it is almost always dephosphorylated. The rate constants were collated to formulate a kinetic model for CheZ-mediated dephosphorylation that includes autoregulation by CheYp and allowed prediction of CheZ activities at CheZ and CheYp concentrations likely to be present in cells.     

Kinetic and thermodynamic studies of tet repressor-tetracycline interaction

Stopped-flow measurements have been employed to study the kinetics of the conformational changes in TetR (B) induced by tetracycline binding with and without Mg(2+) ions. Result of stopped-flow fluorometry measurements at pH 8.0 indicate conformational changes in the helix-turn-helix motif in the N-terminal domain and in the C-terminal inducer binding domain. Binding of tetracycline (Tc) to TetR in the absence of Mg(2+) can be described by a simple kinetics process, which is limited to the first step association without any unimolecular conformational change step upon Tc binding. The rate constants for this process are equal to 2.0 x 10(5) M(-)(1) s(-)(1) and 2.1 s(-)(1) for the forward and backward reaction, respectively, and gave the binding constant K(a) = 0.96 x 10(5) M(-)(1). The kinetics of [Tc-Mg](+) binding to TetR can be described by reactions in which the first step describes the association characterized by the rate constants k(a) = 1.4 x 10(5) M(-)(1) s(-)(1) and k(d) = 2.2 x 10(-)(2) s(-)(1) and binding constant K(a) = 6.3 x 10(6) M(-)(1). The first step of [Tc-Mg](+) association is followed by at least three conformational change steps, which occur in the inducer binding site and then propagate to the surroundings of Trp75 and Trp43 residues. The rate constants for the forward, k(c), and backward, k(-)(c), reaction for each of these conformational steps have been determined. The thermodynamics of the binding of tetracycline with and without Mg(2+) to TetR was investigated by isothermal titration calorimetry (ITC) at pH 8.0 and 25 degrees C. The measurement shows that TetR dimer possesses two equivalent binding sites for tetracycline, characterized by binding constant K(a) = 9.0 x 10(6) M(-)(1) and K(a) = 7.0 x 10(4) M(-)(1) for Tc with and without Mg(2+), respectively. The binding of the inducer to TetR, in the presence and absence of Mg(2+) ion, is an enthalpy-driven reaction characterized by DeltaH = -51 kJ mol(-)(1) and DeltaH = -33 kJ mol(-)(1), respectively. The entropy change, DeltaS, for the interaction in the presence of Mg(2+) is equal to -38.9 J K(-)(1) mol(-)(1), and for the tetracycline alone, it was estimated at -17.6 J K(-)(1) mol(-)(1).     

Binding stoichiometry and kinetics of the interaction of a human anthrax toxin receptor, CMG2, with protective antigen

The protective antigen (PA) moiety of anthrax toxin binds to cellular receptors and mediates entry of the two enzymatic moieties of the toxin into the cytosol. Two PA receptors, anthrax toxin receptor (ATR)/tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2), have been identified. We expressed and purified the von Willebrand A (VWA) domain of CMG2 and examined its interactions with monomeric and heptameric forms of PA. Monomeric PA bound a stoichiometric equivalent of CMG2, whereas the heptameric prepore form bound 7 eq. The Kd of the VWA domain-PA interaction is 170 pm when liganded by Mg2+, reflecting a 1000-fold tighter interaction than most VWA domains with their endogenous ligands. The dissociation rate constant is extremely slow, indicating a 30-h lifetime for the CMG2.PA monomer complex. CMG2 metal ion-dependent adhesion site (MIDAS) was studied kinetically and thermodynamically. The association rate constant (approximately 10(5) m(-1) s(-1)) is virtually identical in the presence or absence of Mg2+ or Ca2+ , but the dissociation rate of metal ion liganded complex is up to 4 orders of magnitude slower than metal ion free complex. Residual affinity (Kd approximately 960 nm) in the absence of divalent metal ions allowed the free energy for the contribution of the metal ion to be calculated as 5 kcal mol(-1), demonstrating that the metal ion-dependent adhesion site is directly coordinated by CMG2 and PA in the binding interface. The high affinity of the VWA domain for PA supports its potency in neutralizing anthrax toxin, demonstrating its potential utility as a novel therapeutic for anthrax.     

Stopped-flow and steady-state study of the diphenolase activity of mushroom tyrosinase

The reaction of mushroom (Agaricus bisporus) tyrosinase with dioxygen in the presence of several o-diphenolic substrates has been studied by steady-state and transient-phase kinetics in order to elucidate the rate-limiting step and to provide new insights into the mechanism of oxidation of these substrates. A kinetic analysis has allowed for the first time the determination of individual rate constants for several of the partial reactions that comprise the catalytic cycle. Mushroom tyrosinase rapidly reacts with dioxygen with a second-order rate constant k(+8) = 2.3 x 10(7) M(-)(1) s(-)(1), which is similar to that reported for hemocyanins [(1.3 x 10(6))-(5.7 x 10(7)) M(-)(1) s(-)(1)]. Deoxytyrosinase binds dioxygen reversibly at the binuclear Cu(I) site with a dissociation constant K(D)(O)()2 = 46.6 microM, which is similar to the value (K(D)(O)()2 = 90 microM) reported for the binding of dioxygen to Octopus vulgaris deoxyhemocyanin [Salvato et al. (1998) Biochemistry 37, 14065-14077]. Transient and steady-state kinetics showed that o-diphenols such as 4-tert-butylcatechol react significantly faster with mettyrosinase (k(+2) = 9.02 x 10(6) M(-)(1) s(-)(1)) than with oxytyrosinase (k(+6) = 5.4 x 10(5) M(-)(1) s(-)(1)). This difference is interpreted in terms of differential steric and polar effects that modulate the access of o-diphenols to the active site for these two forms of the enzyme. The values of k(cat) for several o-diphenols are also consistent with steric and polar factors controlling the mobility, orientation, and thence the reactivity of substrates at the active site of tyrosinase.