Dissociation Rate Constants Of Complexes Research Articles

Inhibition of E. coli RecQ Helicase Activity by Structurally Distinct DNA Lesions: Structure—Function Relationships

DNA helicase unwinding activity can be inhibited by small molecules and by covalently bound DNA lesions. Little is known about the relationships between the structural features of DNA lesions and their impact on unwinding rates and processivities. Employing E.coli RecQ helicase as a model system, and various conformationally defined DNA lesions, the unwinding rate constants kobs = kU + kD, and processivities P = (kU/(kU + kD) were determined (kU, unwinding rate constant; kD, helicase-DNA dissociation rate constant). The highest kobs values were observed in the case of intercalated benzo[a]pyrene (BP)-derived adenine adducts, while kobs values of guanine adducts with minor groove or base-displaced intercalated adduct conformations were ~10-20 times smaller. Full unwinding was observed in each case with the processivity P = 1.0 (100% unwinding). The kobs values of the non-bulky lesions T(6-4)T, CPD cyclobutane thymine dimers, and a guanine oxidation product, spiroiminodihydantoin (Sp), are up to 20 times greater than some of the bulky adduct values; their unwinding efficiencies are strongly inhibited with processivities P = 0.11 (CPD), 0.062 (T(6-4)T), and 0.63 (Sp). These latter observations can be accounted for by correlated decreases in unwinding rate constants and enhancements in the helicase DNA complex dissociation rate constants.

Determination of dissociation kinetics of 188Re(I)-pharmaceuticals by free-ion selective radiotracer extraction

Due to physical decay properties commonly associated with therapeutic radionuclides, 188Re (t1/2 = 16.98 h, Emax = 2.12 MeV) is of high interest for endovascular brachytherapy and endoradiotherapy in general. Rhenium precursors in the low oxidation state +I, such as the organometallic fac-[Re(H2O)3(CO)3]+ are promising lead compounds compared to those with oxidation states +III and +V since they can be prepared under mild conditions and do not tend to reoxidize to oxidation state +VII while multidentate ligands can be attached under substitution of coordinated water molecules. This study comprises the application of the Free-Ion Selective Radiotracer Extraction (FISRE) technique in order to determine dissociation rate constants of complexes bearing the [188Re(CO)3]+-core at tracer levels in vitro with regards to their time-dependent kinetic speciation. As ligands, the tridentate l-histidine as well as the dipeptides l-carnosine (β-alanyl-l-histidine) and glycyl-l-histidine were chosen in order to study the effects of different moieties attached to the primary amine of l-histidine.

Effect of ligand nonplanarity and solvent nature on the kinetic stability of zinc porphyrin complexes

Planarity disturbance in metal porphyrin macrorings produces destabilization of complexes in dimethyl sulfoxide-acetic acid and benzene-acetic acid systems, except for cases where the coordination center is additionally stabilized by endocyclic substituents and/or extra ligands. The first dissociation stage of complexes with N-substituted analogs of porphyrins involves substitution of the acido ligand in the strongly shielded coordination sphere by an electron-donor solvent molecule. The revealed dependences of dissociation rate constants on acid concentration in DMSO, untypical of most metal porphyrins, are explained by a change in the type of the attacking species with changing solvent composition. The dissociation rate constants of complexes in an electron donor solvent can be lower by several orders of magnitude than in a weakly solvating medium.

His374 of wheat endoxylanase inhibitor TAXI‐I stabilizes complex formation with glycoside hydrolase family 11 endoxylanases

Wheat endoxylanase inhibitor TAXI-I inhibits microbial glycoside hydrolase family 11 endoxylanases. Crystallographic data of an Aspergillus niger endoxylanase-TAXI-I complex showed His374 of TAXI-I to be a key residue in endoxylanase inhibition. Its role in enzyme-inhibitor interaction was further investigated by site-directed mutagenesis of His374 into alanine, glutamine or lysine. Binding kinetics and affinities of the molecular interactions between A. niger, Bacillus subtilis, Trichoderma longibrachiatumendoxylanases and wild-type TAXI-I and TAXI-I His374 mutants were determined by surface plasmon resonance analysis. Enzyme-inhibitor binding was in accordance with a simple 1 : 1 binding model. Association and dissociation rate constants of wild-type TAXI-I towards the endoxylanases were in the range between 1.96 and 36.1 x 10(4)m(-1) x s(-1) and 0.72-3.60 x 10(-4) x s(-1), respectively, resulting in equilibrium dissociation constants in the low nanomolar range. Mutation of TAXI-I His374 to a variable degree reduced the inhibition capacity of the inhibitor mainly due to higher complex dissociation rate constants (three- to 80-fold increase). The association rate constants were affected to a smaller extent (up to eightfold decrease). Substitution of TAXI-I His374 therefore strongly affects the affinity of the inhibitor for the enzymes. In addition, the results show that His374 plays a critical role in the stabilization of the endoxylanase-TAXI-I complex rather than in the docking of inhibitor onto enzyme.

Metal Speciation Dynamics in Colloidal Ligand Dispersions

In this work we propose a dynamic metal speciation theory for colloidal systems in which the complexing ligands are localized on the surface of the particles; i.e., there is spatial heterogeneity of binding sites within the sample volume. The differences between the complex formation and dissociation rate constants of complexes in colloidal dispersions and those in homogeneous solutions originate from the differences in kinetic and mass transport conditions. In colloidal systems, when the effective rate of dissociation of the surface complexes becomes fully diffusion controlled, its value is defined via the geometrical parameters of the particle. We assess the extent to which the conventional approach of assuming a homogeneously smeared-out ligand distribution overestimates the lability of surface complexes in colloidal ligand dispersions. The validity of the theory is illustrated by application to binding of lead and cadmium by carboxyl modified latex particles: our approach correctly predicts the formation/dissociation rate constants, which differ by several orders of magnitude from their homogeneous solution counterparts.

Kinetics of iron complexation by dissolved natural organic matter in coastal waters

In coastal waters, where conditions are variable and can change rapidly, the kinetics of iron complexation by NOM is a major factor affecting its speciation. In this study we have examined the kinetics of complexation of ferrous and ferric iron by terrigenous NOM in terms of formation rate constants and dissociation rate constants by employing competitive ligand methods with visible spectrophotometry for determination of the complexed iron. Rate constants for organic ferrous complex formation ranged from 500 to 7.5×10 4 M −1 s −1, and rate constants for complex dissociation ranged from 1×10 −6 to 3.6×10 −3 s −1. Formation rate constants for organic ferric complexes were comparable to those determined for strong iron binding ligands found in the open oceans, and from 2.1×10 5 to 9.6×10 7 M −1 s −1. Ferric complex dissociation data were fitted by a two ligand model, with rate constants for both ligand classes typically higher than those for the strong ligands in the open ocean. Rate constants varied from 2×10 −4 to 4×10 −3 s −1 for the ‘weak’ ligand class, and from 1.0×10 −6 to 1.3×10 −4 s −1 for the ‘strong’ class. All rate constants varied by several orders of magnitude between NOM samples of different origin, reflecting the highly variable composition of these substances. Calculated conditional stability constants for organic complexes with ferric species in seawater, Fe′, were generally similar to those measured in the open oceans. Conditional stability constants for the ferrous complexes were less than those for the ferric complexes by two to four orders of magnitude. There was a weak positive correlation between the conditional stability constants for ferrous and ferric complexes, suggesting that similar functional groups are involved in binding each of the two forms of iron. One of the samples of NOM had a conditional stability constant with Fe′ that was comparable with those for siderophores and similar strong iron binding compounds. These results suggest that in the absence of oxidants, complexes between ferrous iron and NOM may be relatively long-lived. Our work also suggests that organic complexes between iron and terrigenous NOM may be quite strong, and will have a major effect on iron solubility in coastal waters.

Kinetics and Thermodynamics of Complex Formation between FeIII and Two Synthetic Chelators of the Dicatecholspermidine Family

This report deals with a kinetic and thermodynamic study of complex formation between FeIII and a new series of synthetic chelators, 2,3- (FR401) and 3,4-dicatecholspermidine (FR295). For the following data, the first value concerns FR295 and the second FR401. Each ligand undergoes five acid-base dissociations, four of which involve the hydroxy groups of the catechol moieties and the final one the central ammonium group. They occur with average at pK2a = 8.40 and 7.95 for the first two hydroxy groups, pK3a = 12.6 and 12.20 for the two remaining hydroxy groups and pK1a = 5.50 and 4.90 for the ammonium group. Complex formation with both chelators occurs rapidly in acidic media (pH < 2), resulting in the second-order complex formation rate constants k1 = 450 M−1 s−1 and 500 M−1 s−1 and first-order complex dissociation rate constants k−1 = 0.82 s−1 and 0.28 s−1. From these two elementary rates, the complex dissociation constant is determined in acidic media: K1 = 1.82·10−3 M and 5.6·10−4 M. The FeIII complexes also undergo five proton dissociations with marked decreases in the catechol pKa value: ΔpK1a = 1.25 and 1.25, ΔpK2a = 6.60 and 6.50, ΔpK3a = 6.55 and 6.95. From ΔpKa and K1, the affinity of both FR295 and FR401 for FeIII is determined: log β = 29 and 31 with pFe = 22 and 25. This new series of chelators is aimed for antibiotic targeting. It proved efficient in inhibiting Plasmodium falciparum growth and delivered iron ions to Gram-negative bacteria.

Dissociation Kinetics of Aqua[(1SR, 4RS, 7RS, 8SR, 11RS, 14RS)‐5,5,7,12,12,14‐Hexamethyl‐1,4,8,11‐Tetraazacyclotetradecane]Copper(II) (Red) Cation in Strongly Acidic, Aqueous Media

AbstractIn order to investigate the effect of structure variation upon complex dissociation rate constants, the kinetics of the dissociation of (C·rac·5,5,7,12,12,14‐hexamethyl‐1,4,8,11‐tetraazacyclotetradecane)copper(II) (red) cation has been examined spectrophotometrically at 25.0°C in 0.1–5.0 M HNO3. The cleavage of the second copper‐nitrogen bond is proposed as the rate‐determining step for this reaction. The possible pathways for the cleavages of the copper‐nitrogen bonds, the factors influencing the dissociation rates, and the factors affecting the relative importance of each of these possible pathways are considered.

Catecholestrogen binding sites in breast cancer

The binding of 2-hydroxyestrone (20H E 1), a catecholestrogen which is the main end product of the 2-hydroxylation of estrogen, was investigated in breast cancers. 20H E 1-specific bindings were found in the cytosol ( K d = 0.54 ± 0.10 nM) and in the endoplasmic reticulum ( K d = 3.36 ± 1.32 nM). The dissociation rate constants of complexes between [ 3H] 2OH E 1 and cytosol or membrane binding sites were 3.30 h −1 and 8.30 h −1 respectively. Qualitative analysis of [ 3H] 2OH E 1 cytosolic complexes demonstrated a specific binding component with a mol. wt of 330,000 Dallons. Specificity experiments showed that nonestrogenic hormones were unable to compete with 20H E 1 for its binding sites, whereas triphenylethylene derivatives and catecholamines were potent 2OH E 1 competitors. The presence of 20H E 1 specific bindings suggests a potential role of catecholestrogen in breast cancer.

The role of zinc-bound water in liver alcohol dehydrogenase catalysis.

To elucidate the role of zinc-bound water in liver alcohol dehydrogenase catalysis, chelation by 1,10-phenanthroline and 2,2-bipyridine was studied. The rate constants for association of both chelating agents to the active center zinc were pH-dependent with a pKa of 9.2 and preferential binding to a protonated form. The binary complex dissociation rate constants were pH-independent for both chelating agents. In the presence of saturating NAD+, the pKa for the equilibrium binding of 2,2-bipyridine was perturbed to 7.6, similar to the functional group previously shown to be involved in NAD+ binding. The presence of saturating imidazole resulted in pH-independent 2,2-bipyridine binding. These studies provide compelling evidence that the ionizing enzyme functional group involved in coenzyme binding, proton liberation, and conformational states is zinc-bound water. The limiting rate of chelation by 2,2-bipyridine was pH-independent, and no limiting rate was observed in the presence of saturating imidazole. These results indicate that the limiting rate of chelation is due to the rate of dissociation of zinc-bound water. The implications of this regarding the role of zinc in catalytic turnover of liver alcohol dehydrogenase are discussed.

Application of the rotated disk electrode to measurement of copper complex dissociation rate constants in marine coastal samples

Application of the rotated disk electrode to measurement of copper complex dissociation rate constants in marine coastal samples

Animal DNA-dependent RNA polymerases 11. Mechanism of the inhibition of RNA polymerases B by amatoxins

The mechanism of the inhibition of purified calf thymus RNA polymerases B by amatoxins has been studied. Although binding of the enzyme to DNA is not inhibited, initiation of RNA synthesis as well as PP i exchange appear to be inhibited to the same extent as chain elongation. Amatoxins do not affect the affinity of RNA polymerases B for any of the four nucleoside triphosphates. There is no release of DNA or RNA when amatoxins are added to a ternary enzyme—DNA—RNA complex. It appears therefore that amatoxins act primarily by preventing the formation of phosphodiester bonds. A sensitive membrane filter assay for amatoxins—RNA polymerases B complex has been used for equilibrium and kinetic studies of amatoxins and polymerases B interaction. It was demonstrated that the binding of one amatoxin molecule per enzyme B molecule is responsible for the observed inhibition of RNA synthesis. The equilibrium association constant ( K A) is high, in the order of 10 8–10 10 M −1, depending on the temperature and on the ionic strength of the reaction. The rate of dissociation ( k 2) of the complex, which follows first-order kinetics, is slow and is markedly decreased when the temperature is decreased. A decrease of the rate of dissociation is also observed when the ionic strength is increased. The rate of association ( k 1) of amatoxins and enzymes B follows second-order kinetics and is much less sensitive to change in temperature than the rate of dissociation. The ratio k 1 k 2 has been found to agree well with the association equilibrium constant K A over a wide range of temperature, except around 0°C. The driving force for the binding reaction comes from a variation of enthalpy of about −20 kcal · mole −1, whereas the entropy change is about −26 cal · mole −1 · degree −1. The results indicate that electrostatic forces are not important in enzymes B—amatoxin interaction. Hydrogen bonds seem to play an essential role in the stability of amatoxin—enzymes B complex as suggested by the values of the thermodynamic parameters and by the study of the dissociation rate constant of complexes between enzymes B and various amatoxins, differing only in the structure of their hydroxylated isoleucine side-chain. However, the overall conformation of amatoxins is of primer importance for their ability to bind and to inhibit RNA polymerases B. There is a good correlation between the affinity of the different amatoxins for calf thymus RNA polymerases B and their in vivo toxicity. Comparison of the association and dissociation rate constants of α-amanitin and O-methyl-demethyl- γ-amanitin—enzymes B complexes with their in vivo toxicity suggests that the factor which governs the in vivo toxicity is not the association rate, but the dissociation rate of the complex.