Calorimetric demonstration of the potential of molecular crowding to emulate the effect of an allosteric activator on pyruvate kinase kinetics

A method based on isothermal calorimetry is described for the direct kinetic assay of pyruvate kinase. In agreement with earlier findings based on the standard coupled assay system for this enzyme in the presence of a fixed ADP concentration, the essentially rectangular hyperbolic dependence of initial velocity upon phosphoenolpyruvate concentration is rendered sigmoidal by the allosteric inhibitor phenylalanine. This effect of phenylalanine can be countered by including a high concentration of a space- filling osmolyte such as proline in the reaction mixtures. This investigation thus affords a dramatic example that illustrates the need to consider potential consequences of thermodynamic nonideality on the kinetics of enzyme reactions in crowded molecular environments such as the cell cytoplasm.

Thermodynamic non-ideality as an alternative source of the effect of sucrose on the thrombin-catalyzed hydrolysis of peptide p-nitroanalide substrates

The inhibitory effect of sucrose on the kinetics of thrombin-catalyzed hydrolysis of the chromogenic substrate S-2238 (D-phenylalanyl-pipecolyl-arginoyl-p-nitroanilide) is re-examined as a possible consequence of thermodynamic non-ideality-an inhibition originally attributed to the increased viscosity of reaction mixtures. However, those published results may also be rationalized in terms of the suppression of a substrate-induced isomerization of thrombin to a slightly more expanded (or more asymmetric) transition state prior to the irreversible kinetic steps that lead to substrate hydrolysis. This reinterpretation of the kinetic results solely in terms of molecular crowding does not signify the lack of an effect of viscosity on any reaction step(s) subject to diffusion control. Instead, it highlights the need for development of analytical procedures that can accommodate the concomitant operation of thermodynamic non-ideality and viscosity effects.

The influence of substrate kinetics on the microbial community structure in granular anaerobic biomass

The development of a strong, active granular sludge bed is necessary for optimal operation of upflow anaerobic sludge blanket reactors. The microbial and mechanical structure of the granules may have a strong influence on desirable properties such as growth rate, settling velocity and shear strength. Theories have been proposed for granule microbial structure based on the relative kinetics of substrate degradation, but contradict some observations from both modelling and microscopic studies. In this paper, the structures of four granule types were examined from full-scale UASB reactors, treating wastewater from a cannery, a slaughterhouse, and two breweries. Microbial structure was determined using fluorescence in situ hybridisation probing with 16S rRNA-directed oligonucleotide probes, and superficial structure and microbial density (volume occupied by cells and microbial debris) assessed using scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The granules were also modelled using a distributed parameter biofilm model, with a previously published biochemical model structure, biofilm modelling approach, and model parameters. The model results reflected the trophic structures observed, indicating that the structures were possibly determined by kinetics. Of particular interest were results from simulations of the protein grown granules, which were predicted to have slow growth rates, low microbial density, and no trophic layers, the last two of which were reflected by microscopic observations. The primary cause of this structure, as assessed by modelling, was the particulate nature of the wastewater, and the slow rate of particulate hydrolysis, rather than the presence of proteins in the wastewater. Because solids hydrolysis was rate limiting, soluble substrate concentrations were very low (below Monod half saturation concentration), which caused low growth rates. (C) 2003 Elsevier Ltd. All rights reserved.

Enzyme deactivation in fixed bed reactors with michaelis-menten kinetics

Rate expression for enzyme poisoning which are consistent with a Michaelis-Menten main reaction are used to analyze the performance of a fixed bed reactor containing immobilized enzyme. When enzyme deactivation results from the irreversible bonding of a product molecule to an existing substrate-enzyme complex, it is shown that minimum enzyme activity can occur in the interior of the bed, well away from the ends. This suggests that bed sectioning techniques may enable direct evaluation of fundamental poisoning mechanisms.

Fixed bed reactors with catalyst poisoning - first order kinetics.

The long performance of an isothermal fixed bed reactor undergoing catalyst poisoning is theoretically analyzed using the dispersion model. First order reaction with dth order deactivation is assumed and the model equations are solved by matched asymptotic expansions for large Peclet number. Simple closed-form solutions, uniformly valid in time, are obtained.

A finite integral transform technique for solving the diffusion-reaction equation with Michaelis-Menten kinetics

An approximate analytical technique employing a finite integral transform is developed to solve the reaction diffusion problem with Michaelis-Menten kinetics in a solid of general shape. A simple infinite series solution for the substrate concentration is obtained as a function of the Thiele modulus, modified Sherwood number, and Michaelis constant. An iteration scheme is developed to bring the approximate solution closer to the exact solution. Comparison with the known exact solutions for slab geometry (quadrature) and numerically exact solutions for spherical geometry (orthogonal collocation) shows excellent agreement for all values of the Thiele modulus and Michaelis constant.

Substrate-inhibited kinetics with catalyst deactivation in an isothermal CSTR

Analytical expressions are developed for the time-dependent reactant concentration and catalyst activity in an isothermal CSTR with Langmuir-Hinshelwood kinetics of deactivation and reaction. Several parallel and series posioning mechanisms are considered for a reactor which, without poisoning, would operate at a unique steady state. The use of matched asymptotic expansions and abandonment of the usual initial-steady-state assumption give results, valid from startup to final loss of activity, whose accuracy can be improved systematically.

Substrate-inhibited kinetics with catalyst deactivation in an isothermal CSTR. II. Multiple pseudosteady states and reactor failure

Analytical expressions are derived for the time and magnitude of failure of an isothermal CSTR with substrate-inhibited kinetics, caused by slow catalyst deactivation under three types of parallel and series mechanisms. Reactors operating at high space velocity are found to be most susceptible to early failure and poisoning by product is more dangerous than by reactant. The magnitude of the jump across steady states depends solely on the Langmuir-Hinshelwood kinetic parameters and a detailed analysis of reactor behavior during the jump itself is given.

The kinetics of NO and N2O reduction over coal chars in fluidised-bed combustion

This paper presents a comprehensive and critical review of the mechanisms and kinetics of NO and N2O reduction reaction with coal chars under fluidised-bed combustion conditions (FBC). The heterogeneous reactions of NO and N2O with char/carbon surface have been well recognised as the most important processes in reducing both NOx and N2O in situ FBC. Compared to NO-carbon reactions in FBC, the reactions of N2O with chars have been relatively less understood and studied. Beginning with the overall reaction schemes for both NO and N2O reduction, the paper extensively discusses the reaction mechanisms including the effects of active surface sites. Generally, NO- and N2O-carbon reactions follow a series of step reactions. However, questions remain concerning the role of adsorbed phases of NO and N2O, and the behaviour of different surface sites. Important kinetics factors such as the rate expressions, kinetics parameters as well as the effects of surface area and pore structure are discussed in detail. The main factors influencing the reduction of NO and N2O in FBC conditions are the chemical and physical properties of chars, and the operating parameters of FBC such as temperature, presence of CO, O-2 and pressure. It is shown that under similar conditions, N2O is more readily reduced on the char surface than NO. Temperature was found to be a very important parameter in both NO and N2O reduction. It is generally agreed that both NO- and N2O-carbon reactions follow first-order reaction kinetics with respect to the NO and N2O concentrations. The kinetic parameters for NO and N2O reduction largely depend on the pore structure of chars. The correlation between the char surface area and the reactivities of NO/N2O-char reactions is considered to be of great importance to the determination of the reaction kinetics. The rate of NO reduction by chars is strongly enhanced by the presence of CO and O-2, but these species may not have significant effects on the rate of N2O reduction. However, the presence of these gases in FBC presents difficulties in the study of kinetics since CO cannot be easily eliminated from the carbon surface. In N2O reduction reactions, ash in chars is found to have significant catalytic effects, which must be accounted for in the kinetic models and data evaluation. (C) 1997 Elsevier Science Ltd.

Ester prodrugs of a potent analgesic, morphine-6-sulfate: syntheses, spectroscopic and physicochemical properties

The aim of this work is to develop 3-acyl prodrugs of the potent analgesic morphine-6-sulfate (M6S). These are expected to have higher potency and/or exhibit longer duration of analgesic action than the parent compound. M6S and the prodrugs were synthesized, then purified either by recrystallization or by semi-preparative HPLC and the structures confirmed by mass spectrometry, IR spectrophotometry and by detailed 1- and 2-D NMR studies. The lipophilicities of the compounds were assessed by a combination of shake-flask, group contribution and HPLC retention methods. The octanol-buffer partition coefficient could only be obtained directly for 3-heptanoylmorphine-6-sulfate, using the shake-flask method. The partition coefficients (P) for the remaining prodrugs were estimated from known methylene group contributions. A good linear relationship between log P and the HPLC log capacity factors was demonstrated. Hydrolysis of the 3-acetyl prodrug, as a representative of the group, was found to occur relatively slowly in buffers (pH range 6.15-8.01), with a small buffer catalysis contribution. The rates of enzymatic hydrolysis of the 3-acyl group in 10% rat blood and in 10% rat brain homogenate were investigated. The prodrugs followed apparent first order hydrolysis kinetics, with a significantly faster hydrolysis rate found in 10% rat brain homogenate than in 10% rat blood for all compounds. (C) 1998 Elsevier Science B.V. All rights reserved.

Hepatic structure-pharmacokinetic relationships: The hepatic disposition and metabolite kinetics of a homologous series of O-acyl derivatives of salicylic acid

1 The hepatic disposition and metabolite kinetics of a homologous series of O-acyl (acetyl, propionyl, butanoyl, pentanoyl, hexanoyl and octanoyl) esters of salicylic acid (C2SA, C3SA, C4SA, C5SA, C6SA and C8SA, respectively) was determined using a single-pass, in-sills rat liver preparation. 2 The hepatic venous outflow profiles for the parent esters and the generated metabolite, salicylic acid (SA) were analysed by HPLC. Non-parametric moments analysis was used to determine the area under the curve (AUC'), mean transit time (MTT) and normalized variance (CV2) for the parent esters and generated SA. 3 Pregenerated SA ([C-14]-salicylic acid) was injected into each liver with the parent ester to determine its distribution characteristics. 4 The overall recovery of ester plus metabolite was 89% of the ester dose injected and independent of the ester carbon number, suggesting that ester extraction was due to hepatic metabolism to salicylic acid. 5 The metabolite AUC' value increased directly with the lipophilicity of the parent ester (from 0.12 for C2SA to 0.95 for C8SA). By contrast, the parent AUC' decreased with the lipophilicity (from 0.85 for C2SA to zero for C8SA). The metabolite MTT value also showed a trend to increase with the lipophilicity of the parent ester (from 15.72 s for C3SA to 61.97 s for C8SA). However, the parent MTT value shows no significant change across the series. 6 The two-compartment dispersion model was used to derive the kinetic parameters for parent ester, pregenerated SA and generated SA. Consequently, these parameters were used to estimate the values of AUG', MITT and CV2 for the parent ester and metabolite. The moments values obtained using the two-compartment dispersion model show similar trends to the corresponding moments values obtained from the outflow profiles using a non-parametric approach. 7 The more lipophilic aspirin analogues are more confined to the portal circulation after oral administration than aspirin due to their more extensive hepatic elimination avoiding systemic prostacyclin inhibition. Given that aspirin's selectivity as an anti-thrombotic agent has been postulated to be due to selective anti-platelet effects in the portal circulation, the more lipophilic and highly extracted analogues are potentially more selective anti-thrombotic agents than aspirin.

Hepatic disposition and metabolite kinetics of a homologous series of diflunisal esters

The hepatic disposition and metabolite kinetics of a homologous series of diflunisal O-acyl esters (acetyl, butanoyl, pentanoyl, anti hexanoyl) were determined using a single-pass perfused in situ rat liver preparation. The experiments were conducted using 2% BSA Krebs-Henseleit buffer (pH 7.4), and perfusions were performed at 30 mL/min in each liver. O-Acyl esters of diflunisal and pregenerated diflunisal were injected separately into the portal vein. The venous outflow samples containing the esters and metabolite diflunisal were analyzed by high performance liquid chromatography (HPLC). The normalized outflow concentration-time profiles for each parent ester and the formed metabolite, diflunisal, were analyzed using statistical moments analysis and the two-compartment dispersion model. Data (presented as mean +/- standard error for triplicate experiments) was compared using ANOVA repeated measures, significance level P < 0.05. The hepatic availability (AUC'), the fraction of the injected dose recovered in the outflowing perfusate, for O-acetyldiflunisal (C2D = 0.21 +/- 0.03) was significantly lower than the other esters (0.34-0.38). However, R-N/f(u), the removal efficiency number R-N divided by the unbound fraction in perfusate f(u), which represents the removal efficiency of unbound ester by the liver, was significantly higher for the most lipophilic ester (O-hexanoyldiflunisal, C6D = 16.50 +/- 0.22) compared to the other members of the series (9.57 to 11.17). The most lipophilic ester, C6D, had the largest permeability surface area (PS) product (94.52 +/- 38.20 mt min-l g-l liver) and tissue distribution value VT (35.62 +/- 11.33 mL g(-1) liver) in this series. The MTT of these O-acyl esters of diflunisal were not significantly different from one another. However, the metabolite diflunisal MTTs tended to increase with the increase in the parent ester lipophilicity (11.41 +/- 2.19 s for C2D to 38.63 +/- 9.81 s for C6D). The two-compartment dispersion model equations adequately described the outflow profiles for the parent esters and the metabolite diflunisal formed from the O-acyl esters of diflunisal in the liver.

Derivatives of 1,3,5-triamino-1,3,5-trideoxy-cis-inositas versatile pentadentate ligands for protein labeling with Re-186/188. Prelabeling, biodistribution, and X-ray structural studies

The pentadentate H(3)bhci [1,3,5-trideoxy-1,3-bis((2-hydroxybenzyl)amino)-cis-inistol] and its bifunctionalized analogue H(3)bhci-glu-H [1,3,5-trideoxy-1,3-bis((2-hydroxybenzyl)amino)-5-glutaramido-cis-inositol] were synthesized, and their coordination chemistry was investigated with inactive rhenium, with no carrier added Re-188 and with carrier added Re-186. The neutral Re(V) complexes [ReO-(bhci)] and [ReO(bhci-glu-H)] are formed in good yields starting from [ReOCl3(P(C6H5)(3))(2)] or in quantitative yield directly from [(ReO4)-Re-186/188](-) in aqueous solution by reduction with Sn(II) or Sn(0). The X-ray structures of [ReO(bhci)] and [ReO(bhci-glu-H)] were elucidated revealing pentadentate side on coordination of the ligands to the Re=O core. The basic cyclohexane frame adopts a chair form in the case of [ReO(bhci)] and a twisted boat form in the case of [ReO(bhci-glu-H)]. [ReO(bhci)] crystallizes in the monoclinic space group C2/c with a = 27.425(3), b = 14.185(1), c = 19.047(2) Angstrom, and beta = 103.64(2)degrees and [ReO(bhci-glu-H)] in the monoclinic space group P2(1)/c with a = 13.056(3), b = 10.180(1), c = 22.378(5) Angstrom and beta = 98.205(9)degrees Both Re-188 complexes are stable in human serum for at least 3 days without decomposition. After injection into mice, [ReO(bhci-glu)](-) is readily excreted through the intestines, while [ReO(bhci)] is excreted by intestines, liver, and the kidneys. TLC investigations of the urine showed exclusively the complexes [ReO(bhci-glu-H)] and [ReO(bhci)], respectively, and no decomposition products. For derivatization of antibodies, the carboxylic group of [ReO(bhci-glu-H)] was activated with N-hydroxysuccinimide, which required unusually vigorous reaction conditions (heating). The anti colon cancer antibody mAb-35 [IgG and F(ab')(2) fragment] was labeled with [(ReO)-Re-186/188(bhci-glu)] to a specific activity of up to 1.5 mCi/mg (55 MBq/mg) with full retention of immunoreactivity. Labeling yields followed pseudo-first-order kinetics in antibody concentration with the ratio of rates between aminolysis and hydrolysis being about 2. Biodistributions of Re-186-labeled intact mAb-35 as well as of its F(ab')(2) fragment in tumor-bearing nude mice revealed good uptake by the tumor with only low accumulation of radioactivity in normal tissue.