Kinetics Of Dimerization Research Articles

Thermal dimerization kinetics of 3-(p-bromo-phenyl)-pyridazinium benzoyl methylid in solutions

3-(p-Bromo-phenyl)-pyridazinium-benzoyl methylid (BPPBM) participates in solution at 3 + 3 dipolar thermal dimerization that can be spectrally monitored by the extinction in its visible intramolecular charge transfer (ICT) band. The attenuation of ICT band intensity shows the decrease of the BPPBM concentration with the increasing of dimer concentration. The complex kinetics of light-assisted dimerization process was studied taking into account that the thermodynamic equilibrium is reached after more than 24 h. On the basis of general order of reaction theory, we found that the dimerization reaction must be described as a multistep mechanism. The rate constants of the dimerization reactions in ethanol (k = 0.00897 s−1) and benzene (k = 0.00774 s−1) solutions were correlated with the BPPBM and dimer structural features established by using the HyperChem 5.02 simulation program package. A kinetic mechanism of 3 + 3 dipolar thermal dimerization for the studied ylid is proposed. © 2008 Wiley Periodicals, Inc. Int J Chem Kinet 40: 230–239, 2008

Control of diferulate formation in dicotyledonous and gramineous cell-suspension cultures

Primary cell wall polysaccharides of some plants carry ester-linked feruloyl groups that can be oxidatively dimerised both within the protoplast and after secretion into the apoplast. Apoplastic dimerisation has been postulated to form inter-polysaccharide cross-links, contributing to wall assembly, but this role remains conjectural. By feeding cultured cells with [14C]cinnamate, we monitored the kinetics of polysaccharide-binding and subsequent dimerisation of 14C-labelled feruloyl groups. Cultured maize and spinach cells took up [14C]cinnamate more rapidly than barley, Arabidopsis, Acer, tomato and rose cultures. Maize and spinach cells rapidly formed [14C]feruloyl-polysaccharides and, simultaneously, low-Mr [14C]feruloyl esters. When all free [14C]cinnamate had been consumed, there followed a gradual recruitment of radiolabel from the low-Mr pool into the polysaccharide fraction. A proportion of the [14C]feruloyl-polysaccharides was sloughed into the culture medium, the rest remaining wall-bound. Some of the polysaccharide-bound [14C]feruloyl groups were coupled to form dehydrodiferulates. At least six putative isomers of [14C]dehydrodiferulate were formed both rapidly (thus intra-protoplasmically) and gradually (thus mainly apoplastically). These data do not support the hypothesis that intra-protoplasmic dimerisation yields predominantly one isomer (8-5'-dehydrodiferulate). In maize, apoplastic coupling was much more extensive in 7-day old than in 2-day-old cultures; indeed, in 2-day-old cultures apoplastic coupling could not be evoked even by exogenous H2O2, suggesting strong control of peroxidase action by apoplastic factors. When apoplastic coupling was minimised by exogenous application of peroxidase-blockers (iodide, dithiothreitol and cysteine), a higher proportion of the secreted [14C]feruloyl-polysaccharides was sloughed into the medium. This observation lends support to the hypothesis that feruloyl coupling contributes to wall assembly.

Kinetics of UV Light-induced Cyclobutane Pyrimidine Dimers in Human Skin In Vivo: An Immunohistochemical Analysis of both Epidermis and Dermis

Kinetics of UV Light-induced Cyclobutane Pyrimidine Dimers in Human Skin In Vivo: An Immunohistochemical Analysis of both Epidermis and Dermis

Large Propeptides of Fungal β-N-Acetylhexosaminidases Are Novel Enzyme Regulators That Must Be Intracellularly Processed to Control Activity, Dimerization, and Secretion into the Extracellular Environment

Filamentous fungi produce and secrete beta-N-acetylhexosaminidases, Hex, as important components of the binary chitinolytic systems involved in the formation of septa and hyphenation. Enzyme reconstitution experiments published previously indicate that Hex can occur in the form of two molecular species containing either one or two molecules of the propeptide noncovalently associated with the enzyme dimer. Here, we describe a novel mechanism for the regulation of the activity of Hex based on the association of their catalytic subunits with the large N-terminal propeptides in vivo. We show that the enzyme precursor is processed early in the biosynthesis, shortly after the addition of N-glycans through the action of a dibasic peptidase, cleaving both before and after the dibasic sequence. The processing site for this unique dibasic peptidase, different from that of kexins, is conserved among the beta-N-acetylhexosaminidases from filamentous fungi, and inhibition of the dibasic peptidase abrogates enzyme folding and activation. Binding of the released propeptide to the catalytic subunit of Hex is essential for its activation. An examination of the kinetics of Hex activation and dimerization in vitro allowed us to understand the unusually high efficiency of the assembly of this enzyme. We also report that the fungus is able to actively regulate the concentration of the processed propeptide in endoplasmic reticulum and thus the specific activity of the produced Hex. This novel regulatory mechanism enables the control of the catalytic activity and architecture of the secreted enzyme according to the needs of the producing cell at various stages of its growth cycle.

Off-rate and concentration diversity in multidonor-derived dimers of immunoglobulin G

IgG-dimers in multidonor-derived preparations of IgG antibodies represent not only agents of therapeutic potential, but also molecules of basic immunological interest since their composition mirrors the currently unknown range of clonal concentrations and affinities. To analyze this fundamental type of diversity, a computational model is developed in agreement with a density functional theory and used to simulate the dissociation kinetics of dimers separated from a 5000 donor-derived IgG preparation (protein concentration: 0.74mg/mL) via superimposition of 8100 arbitrary combinations of off-rates and initial concentrations. The Greedy algorithm-like procedure described requires iterative and consecutive changes of 8 from a total of 11 fitting parameters and allows to approximate the probability density distributions of either quantities within defined limits (apparent off-rates: ∼4×10−4 to 9×10−17s−1; concentrations: ∼3×10−20 to 1×10−11M) by lognormal distributions of log–log10-type, each of them adapted with four particular parameters, as well as the number of different dimer populations (∼2×1013). Moreover, reasonably dimensioned equilibrium constants involved in monovalent and bivalent random IgG dimerization are estimated by using a mean on-rate of 2.5×105M−1s−1 and interrelationships of molecular parameters derived from known models for antibody–antigen interaction.

Modeling and numerical simulation of biotin carboxylase kinetics: Implications for half-sites reactivity

Biotin carboxylase catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase that catalyzes the first committed step in fatty acid synthesis in all organisms. In Escherichia coli, biotin carboxylase exists as a homodimer where each subunit contains a complete active site. In a previous study (Janiyani, K., Bordelon, T., Waldrop, G.L., Cronan Jr., J.E., 2001. J. Biol. Chem. 276, 29864–29870), hybrid dimers were constructed where one subunit was wild-type and the other contained an active site mutation that reduced activity at least 100-fold. The activity of the hybrid dimers was only slightly greater than the activity of the mutant homodimers and far less than the expected 50% activity for completely independent active sites. Thus, there is communication between the two subunits of biotin carboxylase. The dominant negative effect of the mutations on the wild-type active site was interpreted as alternating catalytic cycles of the active sites in the homodimer. In order to test the hypothesis of oscillating catalytic cycles, mathematical modeling and numerical simulations of the kinetics of wild-type, hybrid dimers, and mutant homodimers of biotin carboxylase were performed. Numerical simulations of biotin carboxylase kinetics were the most similar to the experimental data when an oscillating active site model was used. In contrast, alternative models where the active sites were independent did not agree with the experimental data. Thus, the numerical simulations of the proposed kinetic model support the hypothesis that the two active sites of biotin carboxylase alternate their catalytic cycles.

Kinetic modelling of the liquid-phase dimerization of isoamylenes on Amberlyst 35

Selectivity, conversion, yield and kinetics of the liquid-phase dimerization of 2-methyl-1-butene and 2-methyl-2-butene mixture have been studied in a batch stirred tank reactor in the temperature range 60–100 °C catalysed by the acid resin Amberlyst 35 and using ethanol as a selectivity enhancer. Selectivity to dimers showed a maximum at R IA/EtOH = 20. Obtained diisoamylenes consisted mainly of 3,4,4,5-tetramethyl-2-hexene, 2,3,4,4-tetramethyl-1-hexene and 3,4,5,5-tetramethyl-2-hexene. LHHW type kinetic model was derived for the dimerization reaction as a whole. The kinetic model assumes that three active sites take part in the rate-limiting step of dimerization; the apparent activation energy for the dimerization reaction being 65 kJ mol −1. A pseudohomogeneous kinetic model was used to describe the dimerization reaction network. The apparent activation energy for each dimerization reaction was found to be in the range 61–81 kJ mol −1.

Kinetics, inhibition and oligomerization of Epstein-Barr virus protease

Epstein-Barr virus (EBV) is an omnipresent human virus causing infectious mononucleosis and EBV associated cancers. Its protease is a possible target for antiviral therapy. We studied its dimerization and enzyme kinetics with two enzyme assays based either on the release of paranitroaniline or 7-amino-4-methylcoumarin from labeled pentapeptide (Ac-KLVQA) substrates. The protease is in a monomer–dimer equilibrium where only dimers are active. In absence of citrate the K d is 20 μM and drops to 0.2 μM in presence of 0.5 M citrate. Citrate increases additionally the activity of the catalytic sites. The inhibitory constants of different substrate derived peptides and α-keto-amide based inhibitors, which have at best a K i of 4 μM, have also been evaluated.

Properties of the C-terminal Domain of Enzyme I of the Escherichia coli Phosphotransferase System

The bacterial phosphoenolpyruvate (PEP):glycose phosphotransferase system (PTS) mediates uptake/phosphorylation of sugars. The transport of all PTS sugars requires Enzyme I (EI) and a phosphocarrier histidine protein of the PTS (HPr). The PTS is stringently regulated, and a potential mechanism is the monomer/dimer transition of EI, because only the dimer accepts the phosphoryl group from PEP. EI monomer consists of two major domains, at the N and C termini (EI-N and EI-C, respectively). EI-N accepts the phosphoryl group from phospho-HPr but not PEP. However, it is phosphorylated by PEP(Mg(2+)) when complemented with EI-C. Here we report that the phosphotransfer rate increases approximately 25-fold when HPr is added to a mixture of EI-N, EI-C, and PEP(Mg(2+)). A model to explain this effect is offered. Sedimentation equilibrium results show that the association constant for dimerization of EI-C monomers is 260-fold greater than the K(a) for native EI. The ligands have no detectable effect on the secondary structure of the dimer (far UV CD) but have profound effects on the tertiary structure as determined by near UV CD spectroscopy, thermal denaturation, sedimentation equilibrium and velocity, and intrinsic fluorescence of the 2 Trp residues. The binding of PEP requires Mg(2+). For example, there is no effect of PEP on the T(m), an increase of 7 degrees C in the presence of Mg(2+), and approximately 14 degrees C when both are present. Interestingly, the dissociation constants for each of the ligands from EI-C are approximately the same as the kinetic (K(m)) constants for the ligands in the complete PTS sugar phosphorylation assays.

Kinetics of the ATP Hydrolysis Cycle of the Nucleotide-binding Domain of Mdl1 Studied by a Novel Site-specific Labeling Technique

We have recently proposed a "processive clamp" model for the ATP hydrolysis cycle of the nucleotide-binding domain (NBD) of the mitochondrial ABC transporter Mdl1 (Janas, E., Hofacker, M., Chen, M., Gompf, S., van der Does, C., and Tampé, R. (2003) J. Biol. Chem. 278, 26862-26869). In this model, ATP binding to two monomeric NBDs leads to formation of an NBD dimer that, after hydrolysis of both ATPs, dissociates and releases ADP. Here, we set out to follow the association and dissociation of NBDs using a novel minimally invasive site-specific labeling technique, which provides stable and stoichiometric attachment of fluorophores. The association and dissociation kinetics of the E599Q-NBD dimer upon addition and removal of ATP were determined by fluorescence self-quenching. Remarkably, the rate of ATP hydrolysis of the wild type NBD is determined by the rate of NBD dimerization. In the E599QNBD, however, in which the ATP hydrolysis is 250-fold reduced, the ATP hydrolysis reaction controls dimer dissociation and the overall ATPase cycle. These data explain contradicting observations on the rate-limiting step of various ABC proteins and further demonstrate that dimer formation is an important step in the ATP hydrolysis cycle.

A non-thermal lattice gas model for a dimer–trimer reaction on a catalytic surface: A computer simulation study

The kinetics of an irreversible dimer–trimer reaction of the type 3A2+2B3→6AB have been studied using a non-thermal (precursor mechanism) model on a square as well as on a hexagonal lattice surface by Monte Carlo simulation. When the range of the precursors (A atoms) is increased, the model gives production rates (reactive window widths) that are quite large as compared with those for thermal (Langmuir–Hanshelwood mechanism) model. The phase diagrams qualitatively resemble with the standard ZGB model except that the continuous transition point is eliminated when the range of the precursors is extended up to the third nearest neighbourhood. The diffusion of A atoms on the surface as well as their desorption from the surface with a certain probability is also considered to see their effects on the reaction mechanism.

Folding Kinetics of the S100A11 Protein Dimer Studied by Time-Resolved Electrospray Mass Spectrometry and Pulsed Hydrogen−Deuterium Exchange

This study reports the application of electrospray ionization (ESI) mass spectrometry (MS) with on-line rapid mixing for millisecond time-resolved studies of the refolding and assembly of a dimeric protein complex. Acid denaturation of S100A11 disrupts the native homodimeric protein structure. Circular dichroism and HSQC nuclear magnetic resonance measurements reveal that the monomeric subunits unfold to a moderate degree but retain a significant helicity and some tertiary structural elements. Following a rapid change in solution conditions to a slightly basic pH, the native protein reassembles with an effective rate constant of 6 s(-)(1). The ESI charge state distributions measured during the reaction suggest the presence of three kinetic species, namely, a relatively unfolded monomer (M(U)), a more tightly folded monomeric reaction intermediate (M(F)), and dimeric S100A11. These three forms exhibit distinct calcium binding properties, with very low metal loading levels for M(U), up to two calcium ions for M(F), and up to four for the dimer. Surprisingly, on-line pulsed hydrogen-deuterium exchange (HDX) reveals that each of the monomeric forms of the protein comprises two subspecies that can be distinguished on the basis of their isotope exchange levels. As the reaction proceeds, the more extensively labeled species are depleted. The exponential nature of the measured intensity-time profiles implies that the rate-determining step of the overall process is a unimolecular event. The kinetics are consistent with a sequential folding and assembly mechanism involving two increasingly nativelike monomeric intermediates en route to the native S100A11 dimer.

Conversion, selectivity and kinetics of the liquid-phase dimerisation of isoamylenes in the presence of C 1 to C 5 alcohols catalysed by a macroporous ion-exchange resin

This study evaluated the selectivity, conversion, yield, and kinetics of the liquid-phase dimerisation of 2-methyl-1-butene and 2-methyl-2-butene mixture catalysed by the acid resin Amberlyst 35 in a batch-stirred tank reactor in the temperature range 333–373 K in the presence of 10% mol alcohol content (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, t-butanol, and 1-pentanol). Dimers formation was favoured by increasing temperature and molecular weight of the alcohol, except for methanol. Secondary alcohols slowed dimers formation more than primary alcohols did, and for t-butanol, neither ether nor dimers formation was detected. The obtained isomeric mixture of diisoamylenes consisted mainly of 3,4,4,5-tetramethyl-2-hexene, 2,3,4,4-tetramethyl-1-hexene, and 3,4,5,5-tetramethyl-2-hexene. LHHW- and ER-type kinetic models were derived for the dimerisation reaction, and their parameters were estimated by fitting experimental data. The best kinetic model was that in which three active sites took part in the rate-limiting step of dimerisation, with an apparent activation energy for the dimerisation reaction in the range of 34–53 kJ mol −1.

Effect of Coligands on Biodistribution Characteristics of Ternary Ligand 99mTc Complexes of a HYNIC-Conjugated Cyclic RGDfK Dimer

This report describes biodistribution characteristics of three ternary ligand complexes [(99m)Tc(SQ168)(tricine)(L)] (SQ168 = [2-[[[5-[carboonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-Glu(cyclo{Lys-Arg-Gly-Asp-d-Phe})-cyclo{Lys-Arg-Gly-Asp-d-Phe}; L = TPPTS (trisodium triphenylphosphine-3,3',3' '-trisulfonate), ISONIC (isonicotinic acid) and PDA (2,5-pyridinedicarboxylic acid)) in athymic nude mice bearing MDA-MB-435 human breast cancer xenografts. Ternary ligand complexes [(99m)Tc(SQ168)(tricine)(L)] (L = TPPTS, ISONIC and PDA) were prepared and were analyzed by a reversed HPLC method. Surprisingly, coligands have little impact on log P values of their ternary ligand (99m)Tc complexes even though HPLC retention times suggest that [(99m)Tc(SQ168)(tricine)(PDA)] and [(99m)Tc(SQ168)(tricine)(ISONIC)] are more hydrophilic than [(99m)Tc(SQ168)(tricine)(TPPTS)]. The results from biodistribution studies indicated that excretion kinetics of the (99m)Tc-labeled cyclic RGDfK dimer can be modified by the choice of coligand. The fact that all three radiotracers show high tumor uptake during the 2 h study period suggests that the coligand has minimal effect on the tumor targeting capability of the (99m)Tc-labeled cyclic RGDfK dimer. Results from the blocking experiment suggest that the tumor localization of the (99m)Tc-labeled cyclic RGDfK dimer is integrin alpha(v)beta(3)-mediated. On the basis of their liver uptake and tumor/liver ratios, we believe that PDA has the advantage over TPPTS and ISONIC for the (99m)Tc-labeling of HYNIC-biomolecule conjugates.

Structure, internal motions and association-dissociation kinetics of the i-motif dimer of d(5mCCTCACTCC).

At slightly acidic pH, the association of two d(5mCCTCACTCC) strands results in the formation of an i-motif dimer. Using NMR methods, we investigated the structure of [d(5mCCTCACTCC)]2, the internal motion of the base pairs stacked in the i-motif core, the dimer formation and dissociation kinetics versus pH. The excellent resolution of the 1H and 31P spectra provided the determination of dihedral angles, which together with a large set of distance restraints, improve substantially the definition of the sugar-phosphate backbone by comparison with previous NMR studies of i-motif structures. [d(5mCCTCACTCC)]2 is built by intercalation of two symmetrical hairpins held together by six symmetrical C•C+ pairs and by pair T7•T7. The hairpin loops that are formed by a single residue, A5, cross the narrow grooves on the same side of the i-motif core. The base pair intercalation order is C9•C9+/5mC1•5mC1+/C8•C8+/C2•C2+/T7.T7/C6•C6+/C4•C4+. The T3 bases are flipped out in the wide grooves. The core of the structure includes four long-lived pairs whose lifetimes at 15°C range from 100 s (C8•C8+) to 0.18 s (T7•T7). The formation rate and the lifetime of [d(5mCCTCACTCC)]2 were measured between pH 6.8 and 4.8. The dimer formation rate is three to four magnitude orders slower than that of a B-DNA duplex. It depends on pH, as it must occur for a bimolecular process involving non cooperative association of neutral and protonated residues. In the range of pH investigated, the dimer lifetime, 500 s at 0°C, pH 6.8, varies approximately as 10−pH.

Supraphysiological concentrations of 5-aminolevulinic acid dimerize in solution to produce superoxide radical anions via a protonated dihydropyrazine intermediate

5-Aminolevulinic acid (ALA) is the committed biological precursor to porphyrins. At supraphysiological concentrations ALA can dimerize to form 3,6-dihydropyrazine-2,5-dipropanoic acid (DHPY), which transfers electrons to XTT in a reaction that does not require metal ions and is specifically inhibited by superoxide dismutase. The formation of DHPY from ALA follows dimerization kinetics with a p K of 7.8 ± 0.1. At pH 11.2, DHPY is relatively stable, but when the pH is dropped to 6.0 rapid conversion to 2,5-(β-carboxyethyl)pyrazine occurs via an intermediate with an absorption maximum of 370 nm. Formation of this intermediate is pH-dependent with a p K of 6.0 ± 0.1. These data indicate that ALA dimerizes to produce superoxide from a protonated form of DHPY. The significance of these results with respect to the concentrations of ALA used in photodynamic therapy, and the increased incidence of liver cancer in acute intermittent porphyria, is discussed.

Kinetic Modeling of the Dimerization of Isobutene

The dimerization of isobutene (2-methylpropene) produces diisobutenes (2,4,4-trimethylpentenes) that can be used as high-octane gasoline components. The dimerization kinetics were studied in experiments carried out in a continuous stirred tank reactor at 60−120 °C with different feed concentrations. A commercial ion-exchange resin catalyst was used together with tert-butyl alcohol (2-methyl-2-propanol), which increases the selectivity for diisobutenes by reducing the activity of the sulfonic acid sites of the resin. Langmuir−Hinshelwood-type kinetic models were derived for the dimerization and trimerization reactions, and their parameters were estimated on the basis of the measured data. The only model to represent the data successfully was one where diisobutenes form using two active sites and triisobutenes via diisobutenes using three active sites. This type of model well describes the triisobutene formation rate and the observed maximum in the diisobutene rate. The apparent activation energy was 30 kJ/mol for dimerization.

Dimerization kinetics of thiacarbocyanine dyes by photochemically induced concentration jump

A novel photochemically induced concentration jump method was used to study the fast kinetics of dimerization of thiacarbocyanine dyes in aqueous solution. Perturbation of the monomer–dimer equilibrium is due to fast production of monomers which takes place as a result of an electron transfer reaction photosensitized by a cationic ethyl meso-thiacarbocyanine ( 1) and similar anionic dyes ( 2– 6) in the triplet state. The subsequent restoration of the equilibrium was monitored by changes of monomer absorbance and characterized by a single relaxation time. The kinetics of dimerization were analyzed and the rate constants of dimerization and deaggregation determined.

Trichothiodystrophy fibroblasts are deficient in the repair of ultraviolet-induced cyclobutane pyrimidine dimers and (6-4)photoproducts.

A photosensitive form of trichothiodystrophy (TTD) results from mutations in the same XPD gene as the DNA-repair-deficient genetic disorder xeroderma pigmentosum group D (XP-D). Nevertheless, unlike XP, no increase in skin cancers appears in patients with TTD. Although the ability to repair ultraviolet (UV)-induced DNA damage has been examined to explain their cancer-free phenotype, the information accumulated to date is contradictory. In this study, we determined the repair kinetics of cyclobutane pyrimidine dimers (CPD) and (6-4)photoproducts (6-4PP) in three TTD cell strains using an enzyme-linked immunosorbent assay. We found that all three TTD cell strains are deficient in the repair of CPD and of 6-4PP. UV sensitivity correlated well with the severity of repair defects. Moreover, accumulation of repair proteins (XPB and proliferating cell nuclear antigen) at localized DNA damage sites, detected using micropore UV irradiation combined with fluorescent antibody labeling, reflected their DNA repair activity. Importantly, mutations of the XPD gene affected both the recruitment of the TFIIH complex to DNA damage sites and the TFIIH expression. Our results suggest that there is no major difference in the repair defect between TTD and XP-D and that the cancer-free phenotype in TTD is unrelated to a DNA repair defect.

A NONTHERMAL MODEL FOR CATALYTIC SURFACE REACTION OF THE TYPE A2+B2→2AB: A MONTE CARLO SIMULATION STUDY

The kinetics of irreversible dimer–dimer reaction of the type A2+B2→2AB has already been studied through Monte Carlo simulation via a model based on Langmuir–Hinshelwood (thermal) mechanism. The results of this study are well known. There is single transition point (yC) at yB=0.5 (where yBis partial pressure of B2dimer in gas phase), which separates the two poisoned states from each other. Here, we have studied this reaction on the basis of a nonthermal model, which involves the precursor motion of B2molecule. The most interesting feature of this model is that it yields a steady reactive window. The phase diagram is similar to the ZGB model. The reactive window is separated by continuous and discontinuous irreversible phase transitions. The width of the reactive window depends upon the mobility of the precursors. The dependence of production rate on partial pressure of B2is shown by simple mathematical equations in our model. Some interesting results are observed when reaction between precursors and chemisorbed B atoms is considered.