The reaction of trimethylamine dehydrogenase with trimethylamine

The reductive half-reaction of trimethylamine dehydrogenase with its physiological substrate trimethylamine has been examined by stopped-flow spectroscopy over the pH range 6.0-11.0, with attention focusing on the fastest of the three kinetic phases of the reaction, the flavin reduction/substrate oxidation process. As in previous work with the slow substrate diethylmethylamine, the reaction is found to consist of three well resolved kinetic phases. The observed rate constant for the fast phase exhibits hyperbolic dependence on the substrate concentration with an extrapolated limiting rate constant (klim) greater than 1000 s-1 at pH above 8.5, 10 degrees C. The kinetic parameter klim/Kd for the fast phase exhibits a bell-shaped pH dependence, with two pKa values of 9.3 +/- 0.1 and 10. 0 +/- 0.1 attributed to a basic residue in the enzyme active site and the ionization of the free substrate, respectively. The sigmoidal pH profile for klim gives a single pKa value of 7.1 +/- 0. 2. The observed rate constants for both the intermediate and slow phases are found to decrease as the substrate concentration is increased. The steady-state kinetic behavior of trimethylamine dehydrogenase with trimethylamine has also been examined, and is found to be adequately described without invoking a second, inhibitory substrate-binding site. The present results demonstrate that: (a) substrate must be protonated in order to bind to the enzyme; (b) an ionization group on the enzyme is involved in substrate binding; (c) an active site general base is involved, but not strictly required, in the oxidation of substrate; (d) the fast phase of the reaction with native enzyme is considerably faster than observed with enzyme isolated from Methylophilus methylotrophus that has been grown up on dimethylamine; and (e) a discrete inhibitory substrate-binding site is not required to account for excess substrate inhibition, the kinetic behavior of trimethylamine dehydrogenase can be readily explained in the context of the known properties of the enzyme.     

Scanning molecular sieve chromatography of interacting protein systems. III. Effect of kinetic parameters on the large zone boundary profiles for local equilibration between mobile and stationary phases

Large zone reaction boundary profiles for molecular sieve chromatography as affected by kinetic parameters have been simulated for local equilibration between the mobile and stationary phases. Our studies of monomer-dimer and monomer-tetramer systems indicate that in a slowly equilibrating system, the kinetic controls operating between the mobile and stationary phases contribute most significantly to the overall boundary profile. In a rapidly equilibrating system, however, the kinetic parameters k(ij) and k(ji) operating in the mobile phase are the principal determinants of the reaction boundary, while the kinetic effects of k(ii) and k-(ii) between the mobile and stationary phases are minimal.     

The kinetics of Schiff-base formation during reconstitution of D-serine apodehydratase from Escherichia coli with pyridoxal 5'-phosphate.

Schiff base formation during reconstitution of D-serine dehydratase (Escherichia coli) from its apoenzyme and pyridoxal 5'-phosphate (pyridoxal-P) has been studied by rapid kinetic techniques using absorbance changes at 436 nm. Three distinct reaction phases have been observed. The first is a very rapid change during which pyridoxal-P is initially bound to the apoenzyme. This step has an equilibrium constant of 1500 M-1 and a forward reaction rate of the order of 2.6 x 10(6) M-1 s-1. The second phase shows a first-order rate constant with a value dependent on pyridoxal-P and corresponds to a first-order step with a forward rate constant of 3.04 s-1 interacting with the initial equilibrium. The final phase is a slow first-order reaction, the rate constant of which is approximately 0.01 s-1 and is independent of pyridoxal-P concentration. The active pyridoxal species has been shown to be the free pyridoxal-P as opposed to hemiacetal or hemimercaptal forms.     

Analysis of the Changes In the Proportion of Clustered Labelled Cells In Epidermis

Abstract. A new cell kinetic approach is presented from which the duration of the S and G₂+ M phases can be estimated. the technique involves an analysis of the spatial distribution of labelled cells in sections or sheets of epithelium (i.e. an analysis of clustered labelled cells). the technique is largely independent of the absolute number of labelled cells and hence is not influenced by factors which affect the absolute number of labelled cells. the technique is described and experimental data from dorsal murine skin are presented. the technique has also been simulated mathematically so that the phase durations and their variances could be estimated. the advantages of the technique are: (1) it is technically simple; (2) it provides at least two independent estimates of the phase durations; (3) unlabelled cells need not be counted (compare with LI or PLM analysis); (4) it is independent of variations in the absolute yield of labelled cells, and (5) it is applicable if the LI is low and the S phase is short (where the PLM technique tends to fail).     

高密度脂蛋白体外氧化修饰动力学研究

在体外ＨＤＬ在Ｃｕ＾２＋诱导下可发生氧化修饰,为了探讨体外务浆高密度脂蛋白（ＨＤＬ）氧化修饰中几种产物的动力学改变,用Ｃｕ＾２＋与ＨＤＬ保温２￣２４ｈ,分别观察了ＨＤＬ氧化修饰过程中硫代巴比妥酸反应物质（ＴＢＡＲＳ）,脂氢过氧化物（ＬＯＯＨ）、共轭二烯（ＣＤ）及相对电泳迁移率（ＲＥＭ）等的变化。结果显示,ＬＯＯＨ和ＣＤ两个指标动力学变化相似,呈现延滞期,扩增期和下降期三个时相,而ＴＢＡＲＳ和ＲＥＭ     

Analysis of the changes in the proportion of clustered labelled cells in epidermis

A new cell kinetic approach is presented from which the duration of the S and G2 + M phases can be estimated. The technique involves an analysis of the spatial distribution of labelled cells in sections or sheets of epithelium (i.e. an analysis of clustered labelled cells). The technique is largely independent of the absolute number of labelled cells and hence is not influenced by factors which affect the absolute number of labelled cells. The technique is described and experimental data from dorsal murine skin are presented. The technique has also been simulated mathematically so that the phase durations and their variances could be estimated. The advantages of the technique are: it is technically simple; it provides at least two independent estimates of the phase durations; unlabelled cells need not be counted (compare with LI or PLM analysis); it is independent of variations in the absolute yield of labelled cells, and it is applicable if the LI is low and the S phase is short (where the PLM technique tends to fail).     

Folding, assembly, and stability of the major light-harvesting complex of higher plants, LHCII, in the presence of native lipids

The influence of thylakoid lipids on the association kinetics and thermal stability of the major light-harvesting complex of photosytem II (LHCII) has been studied in vitro. The apoprotein, light-harvesting chlorophyll a/b-binding protein (Lhcb1), can be refolded and complexed with pigments in detergent solution even in the absence of lipids. Two thylakoid lipids, phosphatidyl glycerol and digalactosyl diacylglycerol, are known to interact specifically with LHCII in vivo. Here we show that both of these lipids, as well as monogalactosyl diacylglycerol, stabilize reconstituted LHCII toward thermal denaturation. Two slow kinetic phases are connected with the establishment of energy transfer between chlorophyll b and chlorophyll a and, thus, are thought to reflect the formation of the pigment-protein complex with tightly coupled chlorophylls. The lipids studied here all have the same effect on the rate of complex assembly in vitro and slow these two kinetic phases by the same degree. Both kinetic phases also slow when reactant concentrations are decreased, suggesting that the corresponding reaction step(s) involve(s) pigment binding.     

Organizations in reaction-diffusion systems: Effects of diffusion and boundary conditions

Chemical Organization Theory (COT) has been successfully applied to analyze complex reaction networks where species interact and new species can emerge. The COT has been well studied, but is yet to analyze high dimensional systems dynamics over time equivalent to ordinary differential equations. Moreover, spatial effects, such as diffusion and boundary conditions have also not been considered yet. Here, we extend the COT to cope with reaction-diffusion systems. Thereby we focus on the effects of diffusion and various boundary conditions. In order to demonstrate the effectiveness of our approach, we analyze two models based on partial differential equations, one of which is on HIV virus dynamics.The analysis shows interesting organizational structures when using different ranges of diffusion rates, as well as for Dirichlet and positive Neumann boundary conditions. The advantage of this novel approach is that it is based solely on the model structure (reaction rules) but is independent of kinetic details, such as rate constants. Hence, it copes with high-dimensional systems without the need of numerical simulations, and it can be applied without detailed mathematical knowledge. Our tool is available, without restriction at https://github.com/stephanpeter/orgs-rds.     

Ligand binding and the catalytic reaction of cytochrome caa(3) from the thermophilic bacterium Rhodothermus marinus

The ligand-binding dynamics and the reaction with O(2) of the fully (five-electron) reduced cytochrome caa(3) from the thermohalophilic bacterium Rhodothermus (R.) marinus were investigated. The enzyme is a proton pump which has all the residues of the proton-transfer pathways found in the mitochondrial-like enzymes conserved, except for one of the key elements of the D-pathway, the helix-VI glutamate [Glu(I-286), R. sphaeroides numbering]. In contrast to what has been suggested previously as general characteristics of thermophilic enzymes, during formation of the R. marinus caa(3)-CO complex, CO binds weakly to Cu(B), and is rapidly (k(Ba) = 450 s(-1)) trapped by irreversible (K(Ba) = 4.5 x 10(3)) binding to heme a(3). Upon reaction of the fully reduced enzyme with O(2), four kinetic phases were resolved during the first 10 ms after initiation of the reaction. On the basis of a comparison to reactions observed with the bovine enzyme, these phases were attributed to the following transitions between intermediates (pH 7.8, 1 mM O(2)): R --> A (tau congruent with 8 micros), A --> P(r) (tau congruent with 35 micros), P(r) --> F (tau congruent with 240 micros), F --> O (tau congruent with 2.5 ms), where the last two phases were associated with proton uptake from the bulk solution. Oxidation of heme c was observed only during the last two reaction steps. The slower transition times as compared to those observed with the bovine enzyme most likely reflect the replacement of Glu(I-286) of the helix-VI motif -XGHPEV- by a tyrosine in the R. marinus enzyme in the motif -YSHPXV-. The presence of an additional, fifth electron in the enzyme was reflected by two additional kinetic phases with time constants of approximately 20 and approximately 720 ms during which the fifth electron reequilibrated within the enzyme.     

Scanning molecular sieve chromatography of interacting protein systems. III. Effect of kinetic parameters on the large zone boundary profiles for local equilibration between mobile and stationary phases.

Large zone reaction boundary profiles for molecular sieve chromatography as affected by kinetic parameters have been simulated for local equilibration between the mobile and stationary phases. Our studies of monomer-dimer and monomer-tetramer systems indicate that in a slowly equilibrating system, the kinetic controls operating between the mobile and stationary phases contribute most significantly to the overall boundary profile. In a rapidly equilibrating system, however, the kinetic parameters kij and kji operating in the mobile phase are the principal determinants of the reaction boundary, while the kinetic effects of kii and k-ii between the mobile and stationary phases are minimal.     

Determining the centre of pressure during walking and running using an instrumented treadmill

In this paper, a new method of determining spatial and temporal gait parameters by using centre of pressure (CoP) data is presented. A treadmill is used which was developed to overcome limitations of regular methods for the analysis of spatio-temporal gait parameters and ground reaction forces during walking and running. The design of the treadmill is based on the use of force transducers underneath a separate left and right plate, which together form the treadmill walking surface. The results of test procedures and measurements show that accurate recordings of vertical ground reaction force can be obtained. These recordings enable a separate analysis of vertical ground reaction forces during double support phases in walking, and the analysis of changes in the centre of pressure (CoP) position during subsequent foot placements. From the CoP data, temporal gait parameters (e.g. duration of left/right support and swing phases) and spatial gait parameters (i.e. left/right step lengths and widths) can be derived.     

Apoflavodoxin folding mechanism: an alpha/beta protein with an essentially off-pathway intermediate.

The folding reaction of Anabaena apoflavodoxin has been studied by stopped-flow kinetics and site-directed mutagenesis. Although the urea unfolding equilibrium is two-state, a transient intermediate accumulates during the folding reaction. The intermediate is monomeric, and it is not related to proline isomerization. Unlike many cases where the presence of an intermediate has been detected either by a burst phase or by the curvature, at low urea concentration, of the otherwise only observable kinetic phase, two kinetic phases are observed in apoflavodoxin folding whose total amplitude equals the amplitude of unfolding. To determine the role of the intermediate in the folding reaction, the apoflavodoxin kinetic data have been fitted to all conceivable three-species kinetic models (either linear or triangular). Using a stepwise fitting procedure, we find that the off-pathway mechanism explains most of the kinetic data (not a slow unfolding phase), the on-pathway mechanism being rejected. By using global analysis, good overall agreement between data and fit is found when a triangular mechanism is considered. The fitted values of the microscopic constants indicate that most of the unfolded molecules refold from the denatured state. Apoflavodoxin thus folds via a triangular, but essentially off-pathway, mechanism. We calculate that the retardation of the folding caused by the off-pathway intermediate is not large. Some unusual properties of the intermediate are discussed.     

Internal electron transfer in cytochrome c oxidase: evidence for a rapid equilibrium between cytochrome a and the bimetallic site.

Internal electron-transfer reactions in cytochrome oxidase following flash photolysis of the CO compounds of the enzyme reduced to different degrees (2-4 electron equiv) have been followed at 445, 605, and 830 nm. Apart from CO dissociation and recombination, two kinetic phases are seen both at 445 and at 605 nm with rate constants of 2 x 10(5) and 1.3 x 10(4) s-1, respectively; at 605 nm, an additional phase with a rate constant of 400 s-1 is resolved. At 830 nm, only the second reaction phase (rate constant of 1.3 x 10(4) s-1) is observed. The amplitude of the first phase is largest with the two-electron-reduced enzyme, whereas that of the second phase is maximal at the three-electron-reduction level. Neither phase shows any marked pH dependence. The reaction in the first phase has a free energy of activation of 41 kJ mol-1 and an entropy of activation of -14 JK-1 mol-1. Analysis suggests that the two rapid reaction phases represent internal electron redistributions between the bimetallic site and cytochrome a, and between cytochrome a and CuA, respectively. The slow phase (400 s-1) probably involves a structural rearrangement.     

Kinetic studies of the folding of heterodimeric monellin: evidence for switching between alternative parallel pathways

Determining whether or not a protein uses multiple pathways to fold is an important goal in protein folding studies. When multiple pathways are present, defined by transition states that differ in their compactness and structure but not significantly in energy, they may manifest themselves by causing the dependence on denaturant concentration of the logarithm of the observed rate constant of folding to have an upward curvature. In this study, the folding mechanism of heterodimeric monellin [double-chain monellin (dcMN)] has been studied over a range of protein and guanidine hydrochloride (GdnHCl) concentrations, using the intrinsic tryptophan fluorescence of the protein as the probe for the folding reaction. Refolding is shown to occur in multiple kinetic phases. In the first stage of refolding, which is silent to any change in intrinsic fluorescence, the two chains of monellin bind to one another to form an encounter complex. Interrupted folding experiments show that the initial encounter complex folds to native dcMN via two folding routes. A productive folding intermediate population is identified on one route but not on both of these routes. Two intermediate subpopulations appear to form in a fast kinetic phase, and native dcMN forms in a slow kinetic phase. The chevron arms for both the fast and slow phases of refolding are shown to have upward curvatures, suggesting that at least two pathways each defined by a different intermediate are operational during these kinetic phases of structure formation. Refolding switches from one pathway to the other as the GdnHCl concentration is increased.     

Transient intermediates in the folding of dihydrofolate reductase as detected by far-ultraviolet circular dichroism spectroscopy

The kinetics of the reversible folding and unfolding of Escherichia coli dihydrofolate reductase have been studied by stopped-flow circular dichroism in the peptide region at pH 7.8 and 15 degrees C. The reactions were induced by concentration jumps of a denaturant, urea. The method can detect various intermediates transiently populated in the reactions although the equilibrium unfolding of the protein is apparently approximated by a two-state reaction. The results can be summarized as follows. (1) From transient circular dichroism spectra measured as soon as the refolding is started, a substantial amount of secondary structure is formed in the burst phase, i.e., within the dead time of stopped-flow mixing (18 ms). (2) The kinetics from this burst-phase intermediate to the native state are multiphasic, consisting of five phases designated as tau 1, tau 2, tau 3, tau 4, and tau 5 in increasing order of the reaction rate. Measurements of the kinetics at various wavelengths have provided kinetic difference circular dichroism spectra for the individual phases. (3) The tau 5 phase shows a kinetic difference spectrum consistent with an exciton contribution of two aromatic residues in the peptide CD region. The absence of the tau 5 phase in a mutant protein, in which Trp 74 is replaced by leucine, suggests that Trp 74 is involved in the exciton pair and that the tau 5 phase reflects the formation of a hydrophobic cluster around Trp 74. From the similarity of the kinetic difference spectrum to the difference between the native spectra of the mutant and wild-type proteins, it appears that Trp 47 is the partner in the exciton pair and that the structure formed in the tau 5 phase persists during the later stages of folding. (4) The later stages of folding show kinetic difference spectra that can be interpreted by rearrangement of secondary structure, particularly the central beta sheet of the protein. The pairwise similarities in the spectrum between the tau 3 and tau 4 phases, and between the tau 1 and tau 2 phases, also suggest the presence of two parallel folding channels for refolding. (5) The unfolding kinetics show three to four phases and are interpreted in terms of the presence of multiple native species. The total ellipticity change in kinetic unfolding reaction, however, agrees with the ellipticity difference between the native and unfolding states, indicating the absence of the burst phase in unfolding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular modeling approach to elucidate gas phase hydrodeoxygenation of guaiacol over a Pd(111) catalyst within DFT framework

Excessive amounts of oxy-functional groups in unprocessed bio-oil vitiate its quality as fuel; therefore, it has to be channelized to upgrading processes, and catalytic hydrodeoxygenation is one of the most suitable routes for the upgrading of crude bio-oil. In this computational work, catalytic hydrodeoxygenation (HDO) of guaiacol, which is an important phenolic compound of crude bio-oil, has been carried out using density functional theory (DFT) over a Pd(111) catalyst. The Pd(111) catalyst surface does not endorse direct eliminations of functional groups of guaiacol; however, it is found to perform excellently in stepwise dehydrogenation reactions of oxy-functionals of guaiacol according to present DFT results. The catechol product, formed through dehydrogenation of the methoxy group, followed by elimination of CH₂ and association of the hydrogen atom, has been identified as one of the major products. The overall reaction rate is controlled by scission of CH₂ from 2-methylene-oxy-phenol with an activation energy demand of 23.06 kcal mol^–1. Further, the kinetic analysis of each reaction step involved in HDO of guaiacol over the Pd(111) catalyst surface has also been carried out at atmospheric pressure and at a wide range of temperatures from 473 to 673 K, with temperature intervals of 50 K. In the kinetic analysis part, various kinetic parameters, such as forward and reverse reaction rate constants, Arrhenius constants, and equilibrium rate constants, are reported. The kinetic modeling of the dominating reaction steps has revealed that even a lower temperature of 473 K provides a favorable reaction environment; and the temperature increment further improves the reaction favorability.     

Transient kinetics and intermediates formed during the electron transfer reaction catalyzed by Candida albicans estrogen binding protein

The transient kinetics of the reaction of the estrogen binding protein (EBP1) from Candida albicans in which hydride is transferred from NADPH to trans-2-hexenal (HXL) in two half-reactions were analyzed using UV-visible spectrophotometric and fluorometric stopped-flow techniques. The simplest model of the first half-reaction involves four steps including very rapid, tight binding (K(d) 相似文献   

Multiparticle computer simulation of photosynthetic electron transport in a thylakoid membrane

A method for multiparticle computer simulation of photosynthetic electron transport in a thylakoid membrane has been developed. The basic principles of this method were described previously. The method is used to describe the cyclic electron flow around photosystem I. The effects of size and shape of the reaction volume on the kinetics of interaction of a mobile carrier with a protein complex and the limited diffusion of reactants were studied. It was shown that the kinetic parameters of photosynthetic electron transport processes depend on the distribution of protein complexes in the membrane. It was shown that the limited nature of diffusion of plastoquinone molecules in the membrane leads to a tenfold decrease in the efficient diffusion coefficient. It was shown that the occurrence of two phases of dark reduction of photooxidized P700+ is due to a heterogeneous spatial organization of the thylakoid membrane of a chloroplast.     

Integrated steady state rate equations and the determination of individual rate constants.

Integrated steady state rate equations have been used to determine the kinetic constants (Vs, Ks, Vp, and Kp) and rate constants (k1, k2, k3, and k4) of the reversible enzyme mechanism: (see article). The fumarase reaction has been used as a model to illustrate the procedures for determining these constants. In contrast to initial velocity studies, the values of the constants have been obtained by examining the enzyme reaction in only one direction rather than in both forward and reverse directions. To accomplish this, a new procedure is described for fitting data to integrated rate equations which eliminates problems encountered when data are analyzed graphically. The advantages of examining on enzyme reaction in one direction with these new procedures allow this method to be extended to the examination of enzymes with simple mechanisms where initial velocities are difficult to measure because either the substrate or product is not readily available, or because the reaction is not readily reversible.     

Kinetic inactivation study of mushroom tyrosinase: intermediate detection by denaturants

The unfolding and inhibition study of mushroom tyrosinase have been studied in the presence of different denaturants such as sodium dodecyl sulfate (SDS), guanidine hydrochloride (GdnHCl), and urea. The kinetic two-phase rate constants were commonly measured from semilogarithmic plots of the activity versus time, which resolved into two straight lines, indicating that the inactivation process consisted of fast and slow phases as a first-order reaction. This result also implied that transient partially folded intermediate existed during tyrosinase unfolding pathway. Mushroom tyrosinase had different behaviors to denaturants in regard with: noncooperative binding manner by SDS while cooperative interactions by GdnHCl and urea; in equilibrium state, SDS-micelle never completely inactivated enzyme activity while GdnHCl has single step denaturation and urea induced a typical transition-like process. Various kinetic parameters for each denaturant were calculated and the possible unfolding pathway scheme was discussed.