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At least 811 records · Page 45Linked to original sources
PubMed1997

Guanine-nucleotide binding and hydrolyzing kinetics of ORrab2, a rice small GTP-binding protein expressed in Escherichia coli.

The ORrab2 gene encodes a GTP-binding protein of 23.169 kDa. The deduced amino acid sequence shows that ORrab2 has the motifs conserved among small GTP-binding proteins in plants and that it shares sequence identity with Atrab2 (93.0%), Hrab2 (85.2%), Hrab4 (51.9%), Hrab1 (46.2%), YPT (40.7%), Hrab3B (40.0%), Hrab3A (38.1%), SEC4 (38.1%), Hrab5 (34.3%) and Hrab6 (32.4%). To analyze the biochemical properties of this protein, an ORrab2 cDNA was overexpressed in Escherichia coli and the protein purified by Ni2+-nitrilotriacetic acid agarose and hydroxyapatite column chromatography. The molecular mass of the protein bearing a His-tag is approximately 28.2 kDa. The guanine-nucleotide binding and hydrolyzing activity of ORrab2 increased with non-ionic C12E10 (polyoxyethylene 10-lauryl ether) and ionic Chaps detergent treatment. ORrab2 bound maximally 1.03 mol of [gamma-35S]GTP[S]/mol of protein with a Kd value of 56.83 nM. The ratios k(off GDP)/k(off GTP) of ORrab2 were 3.63 for the control, 3.7 in the presence of C12E10, and 3.83 with Chaps, indicating that ORrab2 has a higher affinity for GTP than GDP. The rate (k(cat)) of Pi release against [gamma-32P]GTP bound ORrab2 in a steady state and the rate of hydrolysis of [gamma-32P]GTP (kGTPase) were calculated to be 432 x 10(-4) +/- 8 x 10(-4) min(-1) and 172 x 10(-4) +/- 2 x 10(-4) min(-1), respectively, in the presence of 0.1% C12E10 and 1 mM MgSO4.

Amino Acid Sequence↗
PubMed2000

Agonist-induced isomerization in a glutamate receptor ligand-binding domain. A kinetic and mutagenetic analysis.

Agonist binding to glutamate receptor ion channels occurs within an extracellular domain (S1S2) that retains ligand affinity when expressed separately. S1S2 is homologous to periplasmic binding proteins, and it has been proposed that a Venus flytrap-style cleft closure triggers opening of glutamate receptor ion channels. Here we compare the kinetics of S1S2-agonist binding to those of the periplasmic binding proteins and show that the reaction involves an initial rapid association, followed by slower conformational changes that stabilize the complex: "docking" followed by "locking." The motion detected here reflects the mechanism by which the energy of glutamate binding is converted into protein conformational changes within S1S2 alone. In the intact channel, these load-free conformational changes are harnessed and possibly modified as the agonist binding reaction is used to drive channel opening and subsequent desensitization. Using mutagenesis, key residues in each step were identified, and their roles were interpreted in light of a published S1S2 crystal structure. In contrast to the Venus flytrap proposal, which focuses on motion between the two lobes as the readout for agonist binding, we argue that smaller, localized conformational rearrangements allow agonists to bridge the cleft, consistent with published hydrodynamic measurements.

Amino Acid Substitution↗
PubMed1990

Kinetic study of interaction between [14C]amphotericin B derivatives and human erythrocytes: relationship between binding and induced K+ leak.

The relationship between polyene antibiotic binding to red cells and their membrane permeabilization was studied using two 14C-labelled amphotericin B (AmB) derivatives: N-fructosyl AmB and N-acetyl methyl ester AmB. The binding kinetics of both derivatives were determined on whole red cells and ghosts. The resulting experimental points were well fitted by monoexponential functions, and the characteristic t1/2 for both derivatives were calculated from these functions. At 2 X 10(-5) M, the half time t1/2 for N-acetyl methyl ester AmB (30.2 min) which forms aqueous aggregates was longer than the t1/2 for the more soluble species N-fructosyl AmB (4.5 min). At lower concentrations (10(-7) M), the t1/2 for N-acetyl methyl ester AmB (6.3 min) in a more solubilized form was close to that of N-fructosyl AmB (7.9 min). These results suggest that only solubilized species bound to red cell membranes and that disaggregation of aggregates is the limiting step in the binding process. The permeabilization of red cell membranes by N-fructosyl AmB, measured as intracellular K+ leak, was not instantaneous and at 10 degrees C external K+ was only detected 20 min after antibiotic addition. In contrast, binding occurs without lag time. Consequently, different mecanisms underlie binding and K+ permeability inducement. Absorption spectroscopy data showed that bound antibiotic is located in the hydrophobic membrane interior and that this penetration of the membrane by AmB derivatives occurs without lag time. Consequently, the lag time occurring for K+ permeability inducement would be due to some steps subsequent to binding and probably located in the hydrophobic membrane interior. This statement is further supported by the observation that the lag time is sensitive to changes in membrane fluidity as shown here by the break between 20 and 30 degrees C in the slope of the Arrhenius plot for the lag time, coinciding with the phase transition in red cell membranes.

Amphotericin B↗
PubMed1983

Dynamic evidence for an extended subsite structure of the ligand combining site on wheat germ agglutinin: temperature-jump relaxation with fluorescence detection.

Temperature-jump relaxation methods have been used to study the binding kinetics of fluorescent 4-methylumbelliferyl glycosides of N-acetyl-beta-D-glucosamine and its beta (1 leads to 4)-linked di- and trisaccharides with wheat germ agglutinin. The mono- and disaccharide derivatives yielded biexponential progress curves. The data are consistent with two simple mechanisms in which binding occurs to an extended combining site on the lectin, consisting of at least two different, mutually exclusive, binding subsites. For one model, the bound ligand must slide from one subsite to the other, and the other mechanism requires the dissociation of the bound ligand from the protein before it can combine to the other subsite. Binding of 4-methylumbelliferyl monosaccharide to nonequivalent sites is improbable. The underlying kinetic and equilibrium parameters were obtained for the proposed subsites. The binding kinetics of the 4-methylumbelliferyl trisaccharide derivative are more complicated and may result from ligand-mediated linking reactions between molecules of the lectin. This study emphasizes that binding studies at equilibrium should take into account that the data result from an average of different binding configurations of all the ligands.

Acetylglucosamine↗
PubMed2003

Interpreting enzyme and receptor kinetics: keeping it simple, but not too simple.

The hyperbolic parabola is commonly used to summarize kinetics for enzyme reactions and receptor binding kinetics. Depending on the experimental conditions, certain assumptions are valid but others might be violated so that the parameters used to fit this hyperbolic function, the maximum asymptote and the equilibrium constant, require different interpretations. The first topic of this review compares enzyme-induced transformations and receptor binding in terms of the appropriate assumptions. The second topic considers the complication of adding a competitive inhibitor as an enzyme substrate or a receptor ligand and the subtleties of inferring the equilibrium dissociation constant from the concentration of inhibitor (for example unlabeled drug) that leads to the midpoint, IC(50,) of an inhibition curve. Receptor binding may be measured directly by a concentration assay or as a pharmacodynamic response variable.

Binding Sites↗
PubMed1997

[3H]2-(2-Benzofuranyl)-2-imidazoline, a highly selective radioligand for I2-imidazoline receptor binding sites. Studies in rabbit kidney membranes.

2-(2-Benzofuranyl)-2-imidazoline (2-BFI) has recently been characterised as a selective ligand for the I2-type of imidazoline-receptor binding site(s) (I2-RBS). The present studies determined the relative levels of specific [3H]2-BFI binding to membrane homogenates of brain and kidney from rat, guinea pig and rabbit and identified the pharmacological characteristics of [3H]2-BFI binding sites in rabbit kidney membranes. Rabbit kidney membranes had the highest relative density of specific [3H]2-BFI binding of all tissues studied (2000 fmol/mg protein). Rabbit brain and guinea pig kidney had moderate levels of specific [3H]2-BFI binding (350-500 fmol/mg protein), while rat kidney and guinea pig and rat brain displayed much lower densities of binding (40-65 fmol/mg protein). Studies of [3H]2-BFI binding kinetics in rabbit kidney homogenates revealed binding to two distinct sites with Kd values of 0.10 +/- 0.01 nmol/l and 1.00 +/- 0.36 nmol/l respectively. Equilibrium saturation studies were also consistent with the presence of two binding sites-[3H]2-BFI (0.01-20 nmol/l) bound to sites with affinities of 0.10 +/- 0.01 nmol/l and 0.92 +/- 0.13 nmol/l and binding densities of 470 +/- 80 and 840 +/- 60 fmol/mg protein (n = 3), representing 36 and 64% respectively. Drug inhibition studies revealed that L-adrenaline; alpha 1-adrenoceptor drugs (prazosin, L-phenylephrine) and alpha 2-adrenoceptor drugs (rauwolscine, methoxyidazoxan, 2-(2,4-(O-methoxyphenyl)-piperazin-1-yl)-ethyl-4, 4-dimethyl-1,3-(2H,4H)-isoquinolindione (ARC-239) had extremely low affinities for [3H]2-BFI binding sites (IC50 > or = 10 mumol/l). Putative I1-RBS compounds, p-aminoclonidine, moxonidine, imidazole-4-acetic acid and cimetidine, inhibited [3H]2-BFI binding to rabbit renal membranes with low to very low affinities (Ki values 3 to > or = 100 mumol/l), suggesting [3H]2-BFI does not label I1-RBS in rabbit kidney membranes. I2-RBS compounds-2- (4,5-dihydroimidaz-2-yl)-quinoline (BU224), 2-(4,5-dihydroimidaz-2-yl)-quinoxaline (BU239), idazoxan and cirazoline-potently inhibited [3H]2-BFI binding (Ki values 0.37-1.6 nmol/l), confirming the labelling of I2-RBS. Inhibition of [3H]2-BFI binding by certain compounds was consistent with their interaction with two binding site populations-for example (drug, Ki values) guanabenz, 0.65 nmol/l and 0.17 mumol/l; naphazoline, 0.94 nmol/l and 2.8 mumol/l; amiloride, 76 nmol/l and 26 mumol/l rilmenidine, 150 nmol/l and 50 mumol/l; and clonidine, 230 nmol/l and 70 mumol/l. The high affinity of amiloride for a high proportion (85%) of the binding is consistent with the presence of the I2A-subtype of I-RBS in rabbit kidney. These results demonstrate that [3H]2-BFI is a highly selective and high affinity radioligand for I2-RBS which should be useful for the further characterisation of these sites in mammalian tissues.

Animals↗
PubMed1994

Estradiol reduction of the agonist high affinity form of the alpha 2-adrenoceptor in the hypothalamus of female rats: identification as the alpha 2D subtype.

These studies examined which alpha 2-adrenoceptor subtype is expressed in the hypothalamus and preoptic area and the influence of estradiol administration on alpha 2-adrenoceptors in the hypothalamus of female rats. The alpha 2-adrenoceptor antagonist [3H] RX821002 bound to a single site in hypothalamus, preoptic area, and cortex membranes, with high affinity and low nonspecific binding, as determined by Scatchard and kinetic binding analyses. Competition for [3H]RX821002 binding in the hypothalamus and preoptic area by various noradrenergic agonists and antagonists revealed a unique pharmacological specificity with a high degree of similarity to that of the alpha 2D-adrenoceptor. Norepinephrine displacement of [3H]RX821002 binding in hypothalamic membranes from ovariectomized animals was monophasic and characterized by high affinity. In contrast, norepinephrine competition for [3H]RX821002 binding sites in the hypothalamus from rats exposed to estradiol for 48 hr was biphasic, and norepinephrine bound to both a high (18%) and a low (82%) affinity site in these membranes. Thus, the formation of agonist high affinity alpha 2D-adrenoceptor complexes was inhibited by prior exposure to estrogen. In both control and estradiol-exposed hypothalamic membranes, 100 microM 5'-guanylylimidodiphosphate [Gpp(NH)p] converted the norepinephrine competition curves to ones characterized by monophasic, low affinity binding. In addition, binding of the full alpha 2-adrenoceptor agonist [3H]UK-14,304 in the hypothalamus and preoptic area of female rats was concentration-dependently diminished by Gpp(NH)p treatment. Complete loss of [3H]UK-14,304 binding was effected by 100 microM Gpp(NH)p. This suggests that [3H]UK-14,304 may be useful in labeling the agonist high affinity state of alpha 2-adrenoceptors. Decreasing the incubation temperature in saturation studies from 25 degrees to 0 degrees increased [3H]UK-14,304 binding in hypothalamic membranes of control rats but not in membranes from estradiol-treated rats. Estradiol treatment for 48 hr decreased [3H]UK-14,304 binding in hypothalamic membranes by 34% (0 degrees) to 60% (25 degrees), without changing the Kd. These results suggest that the alpha 2D-adrenoceptor is the predominant subtype in the hypothalamus and preoptic area of female rats and that estradiol treatment markedly reduces the number of alpha 2D-adrenoceptors in the agonist high affinity state.

Adrenergic alpha-Agonists↗
PubMed1988

Importance of acyl-CoA availability in interpretation of carnitine palmitoyltransferase I kinetics.

Bovine serum albumin is generally employed as a substrate depot for the delivery of acyl units to lipid metabolizing enzymes in vitro. Here we test the possibility that albumin alters the availability of substrate to mitochondrial carnitine palmitoyltransferase I and thereby alters its apparent kinetics. Binding competition with palmitoyl-CoA indicates that albumin has 5-6 high affinity sites which avidly bind the substrate, while isolated mitochondria compete favorably for substrate only as the albumin sites become saturated. In contrast to albumin, artificial phospholipid vesicles bind palmitoyl-CoA uniformly. Palmitoyl-CoA distribution between vesicles and mitochondrial membranes appears simply to be a function of the relative size of the two lipid compartments. Both albumin and artificial vesicles reduce the effective concentration of substrate available to the enzyme and in this way reduce apparent affinity. Direct measurement of mitochondrially bound substrate removes this effect and brings the results into agreement with an affinity constant of 6-7 nmol/mg. Changes in gross mitochondrial structure, as indicated by decreased optical density and increased nonpelleting protein, do not begin occurring until levels of mitochondrially bound palmitoyl-CoA are 15 times greater than this. The highly sigmoidal activity profile of carnitine palmitoyltransferase with respect to palmitoyl-CoA (apparent Hill coefficient = 3.0 +/- 0.3) is lost when vesicles are substituted for albumin, suggesting that albumin binding sites contribute to the sigmoidal kinetics in the range of palmitoyl-CoA studied.

Acyl Coenzyme A↗
PubMed1985

n-Alkanols and halothane inhibit red cell anion transport and increase band 3 conformational change rate.

The effects of halothane and n-alkanols on band 3, the anion-exchange protein of the red cell membrane, have been characterized by radioactive sulfate exchange and equilibrium and kinetic binding of a fluorescent anion transport inhibitor, 4,4'-dibenzamido-2,2'-stilbenedisulfonic acid (DBDS), with fluorescence and stopped-flow techniques. Ethanol, butanol, hexanol, heptanol, octanol, and decanol inhibit radioactive sulfate efflux from red blood cells in a dose-dependent manner with an average Hill coefficient of 1.3 +/- 0.1. Over a 10(4)-fold range of buffer concentrations, the calculated membrane alkanol concentrations at which anion transport rates are reduced by 50% are 100-200 mM. At 100-300 mM membrane concentrations, halothane and the n-alkanols increase the apparent rate of DBDS binding to band 3 2-3-fold. Analysis of kinetic and equilibrium DBDS binding data shows that these drugs increase the rate of the DBDS-induced conformational change in the DBDS-band 3 complex. Equilibrium DBDS binding studies reveal differences between the actions of short-chain alkanols (ethanol and butanol) and those of long-chain alkanols (hexanol and longer). Short-chain alkanols reduce the equilibrium affinity of DBDS for band 3, while long-chain alkanols have no effect on equilibrium DBDS binding. The results for halothane and long-chain alkanols suggest a nonspecific, lipid-mediated mechanism of anesthetic action, which may be coupled to protein inactivation by an increase in the rate of protein conformational changes resulting in nonfunctional states. The results for short-chain alkanols indicate that they have the same nonspecific actions as the long-chain alkanols but also have specific effects on the stilbene binding site of band 3.

Alcohols↗
PubMed1983

Differential properties of mu and delta opiate binding sites studied with highly selective ligands.

Differences in binding kinetics, ions and nucleotides interactions with rat brain opiate mu and delta receptor subtypes were investigated using respectively DAGO (Tyr-D-Ala-Gly-MePhe-Gly-ol) and DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr) as highly selective mu and delta ligands. On the basis of kinetic experiments the delta-agonist binding is an extremely slow process as compared to that of the mu-agonist. Displacement experiments by DTLET of [3H] DAGO binding and Scatchard analysis of the binding of [3H] DTLET in the absence and presence of DAGO demonstrated that an independent model of interaction to two independent sites best describes the observed data. Steady state binding of [3H] DAGO is decreased by GMP-P(NH)P while this nucleotide is ineffective in reducing the binding of DTLET. However in presence of NaCl, GMP-P(NH)P reduces the specific binding at both sites in a concentration dependent manner. The analysis of the nucleotide inhibition of delta-binding at various concentrations of NaCl suggests that the non-hydrolyzable nucleotide can exerts its effects only on a prebound Na+ receptor. These data suggest that mu and delta-receptors may assume different affinity states depending upon the presence of Na+ and nucleotide.

Animals↗
PubMed1999

The salt dependence of DNA recognition by NF-kappaB p50: a detailed kinetic analysis of the effects on affinityand specificity.

The binding kinetics of NF-kappaB p50 to the Ig-kappaB site and to a DNA duplex with no specific binding site were determined under varying conditions of potassium chloride concentration using a surface plasmonresonance biosensor. Association and dissociation rate constants were measured enabling calculation of the dissociation constants. Under previously established high affinity buffer conditions, the k a for both sequences was in the order of 10(7) M-1s-1whilst the k d values varied 600-fold in a sequence-dependent manner between 10(-1) and 10(-4 )s-1, suggesting that the selectivity of p50 for different sequences is mediated primarily through sequence-dependent dissociation rates. The calculated K D value for the Ig-kappaB sequence was 16 pM, whilst the K D for the non-specific sequence was 9.9 nM. As the ionic strength increased to levels which are closer to that of the cellular environment, the binding of p50 to the non-specific sequence was abolished whilst the specific affinity dropped to nanomolar levels. From these results, a mechanism is proposed in which p50 binds specific sequences with high affinity whilst binding non-specific sequences weakly enough to allow efficient searching of the DNA.

DNA↗
PubMed1976

Effect of hepatocarcinogens on the binding of glucocorticoid-receptor complex in rat liver nuclei.

The effects of a number of carcinogens and hepatotoxins on the binding kinetics of the interactions of glucocorticoidcytosol receptor complex with nuclear acceptor sites in rat liver were investigated. Both the apparent sites in rat liver were investigated. Both the apparent concentration of nuclear binding sites and the Kd were significantly diminished following treatment of rats with sublethal doses of the carcinogens aflatoxin B1, diethylnitrosamine, dimethylnitrosamine, thioacetamide, 3'-methyl-4-dimethylaminoazobenzene, 4-dimethylaminoazobenzene, and 3-methylcholanthrene. Treatment with actinomycin D resulted in a slight reduction in the apparent concentration of nuclear acceptor sites but had no effect on the nuclear binding Kd. The hepatotoxic but noncarcinogenic analgesic, acetaminophen, as well as the weakly toxic aflatoxin B1 cognate, aflatoxin B2, were without effect on the kinetics or binding capacity of glucocorticoid-nuclear acceptor site interaction. These experiments suggest that chemically induced alteration of functional glucocorticoid binding sites on chromatin may be involved in the biochemical effects produced in liver by carcinogens of several chemical types. This experimental model may provide a useful approach for further elucidation of early events in carcinogenesis.

Alkylation↗
PubMed1987

Enhanced antigen-antibody binding affinity mediated by an anti-idiotypic antibody.

We previously described the production of four monoclonal antibodies to the beta-adrenergic receptor antagonist alprenolol. One of these antibodies, 5B7 (IgG2a, kappa), was used to raise anti-idiotypic antisera in rabbits. In contrast to the expected results, one of the anti-idiotypic antisera (R9) promotes [125I]iodocyanopindolol (ICYP) binding to antibody 5B7. In the presence of R9, the dissociation constant decreases 100-fold from 20 to 0.3 nM. This increase in binding affinity of antibody 5B7 for ICYP is not observed in the presence of preimmune, rabbit anti-mouse or anti-idiotypic antisera generated to a monoclonal antibody of a different specificity. Furthermore, R9 in the absence of 5B7 does not bind ICYP. The F(ab) fragments of 5B7 and R9 behaved in a similar manner, and the soluble complex responsible for the high-affinity interaction with ICYP can be identified by gel filtration chromatography. The elution position of the complex is consistent with a 5B7 F(ab)-R9 F(ab) dimer, indicating that polyvalency is not responsible for the enhanced ligand binding. Kinetic analysis of ICYP-5B7 binding revealed that the rate of ICYP dissociation from 5B7 in the presence of R9 is approximately 100 times slower than in the absence of R9 [k-1(+R9) = 0.025 min-1 vs. k-1(-R9) = 2.04 min-1], consistent with the 100-fold change in binding affinity of 5B7 for ICYP. The available data best fit a model in which an anti-idiotypic antibody binds at or near the binding site of the idiotype participating in the formation of a hybrid ligand binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Alprenolol↗
PubMedDate not supplied

Specific interactions of synthetic peptides derived from P. falciparum merozoite proteins with human red blood cells.

In the search for strategies which might help in the elucidation of molecular mechanisms involved in the red blood cell (RBC) invasion by P. falciparum merozoites, and with the specific aim of establishing whether synthetic peptides derived from selected parasite proteins bind to human RBCs, 26 different peptides were chemically synthesized and radiolabeled. It was found that the peptides could be grouped, according to their RBC-binding kinetics, into high, medium and low binding activity. A correlation was detected between the high binding activity of a peptide and the presence of either a KEK motif (or its variants LEK or KEL) or a NVXAA (where X is V or Y). Peptides with medium or low binding activities did not possess either of these two consensus sequences. Selective modification of amino acids within the KEK motif diminished their uptake or binding capacity. Competitive inhibition assays of labeled or unlabeled peptide demonstrated a correlation between the presence of KEK or NVXAA motifs and a high binding activity of a peptide. Invasion-inhibition studies showed a direct correlation between a peptide's binding activity and inhibitions of human RBC reinvasion. Other experiments showed that high binding activity peptides show a decreased uptake with related and nonrelated human erythrocytes.

Amino Acid Sequence↗
PubMed2000

Measurement of protein interaction bioenergetics: application to structural variants of anti-sCD4 antibody.

This chapter has described a bioenergetic analysis of the interaction of sCD4 with an IgG1 and two IgG4 derivatives of an anti-sCD4 MAb. The MAbs have identical VH and VL domains but differ markedly in their CH and CL domains, raising the question of whether their antigen-binding chemistries are altered. We find the sCD4-binding kinetics and thermodynamics of the MAbs are indistinguishable, which indicates rigorously that the molecular details of the binding interactions are the same. We also showed the importance of using multiple biophysical methods to define the binding model before the bioenergetics can be appropriately interpreted. Analysis of the binding thermodynamics and kinetics suggests conformational changes that might be coupled to sCD4 binding by these MAbs are small or absent.

Antibodies, Monoclonal↗
PubMed1987

Theoretical characterization of ion channel blockade. Competitive binding to periodically accessible receptors.

Competitive ligand binding to periodically activated or accessible receptors is influenced by the interaction between ligand binding kinetics and the interval of time the binding site is accessible. This interaction produces a paradoxical reduction in bound receptors under certain conditions. A mathematical description of multi-ligand binding to a single binding site is presented for both the continuously and transiently accessible cases. The theoretical results predict paradoxical "agonism" and are consistent with the results of studies of lidocaine and bupivacaine binding to cardiac sodium channels.

Binding, Competitive↗
PubMed1996

Rate constants of sugar transport through two LamB mutants of Escherichia coli: comparison with wild-type maltoporin and LamB of Salmonella typhimurium.

Two LamB (maltoporin) point mutants of Escherichia coli (R8H and Y118F) and wild-type LamB of Salmonella typhimurium were reconstituted into artificial lipid bilayer membranes. Ion transport through wild-type LamB of S. typhimurium and the LamB mutants was inhibited by the addition of carbohydrates of maltose and maltooligosaccharide type in a dose-dependent fashion. The sugar-induced block of the channel function could be used for the study of current noise through the different wild-type and mutant LamB-channels. The analysis of the power density spectra allowed the evaluation of the on and off-reactions (k1 and k-1) of sugar-binding to the binding site inside the channels. Wild-type LamB of S. typhimurium had approximately the same sugar-binding kinetics as has been observed for LamB of E. coli. The results suggest that the binding site inside the channel interacts with a maximum of three glucose residues within the maltooligosaccharides. The LamB mutants R8H and Y118F showed kinetics for sugar binding substantially different from that of wild-type LamB. In particular, the on-rate, k1, for the binding of different sugars of the maltooligosaccharide series to the mutant R8H was approximately 500-times smaller than for wild-type LamB, which resulted in substantially smaller stability constant of sugar binding to the channel. Similarly, the off-rate constant, k-1, for sugar binding to the mutant Y118F decreased about 20-fold, which led to a strong increase of the affinity of carbohydrates to the site. The role of the amino residues acid R8 and Y118 in the transport of maltose and maltooligosaccharides through LamB-channels is discussed on the basis of the net flux of sugars through the channels.

Alkanes↗