Simple Michaelis-Menten Kinetics Research Articles

A combined mathematical model to represent transpiration, respiration, and water activity changes in fresh cape gooseberry (Physalis peruviana) fruits

In this work, a combined respiration and transpiration model is proposed for fresh cape gooseberry fruits, also considering changes in water activity (aw) and moisture content. Initially, a correlation of parameters was obtained from experimental data to determine the kinetics of O2 consumption and CO2 production for cape gooseberry fruits at different temperatures. From the data on the change in gas concentration, a parameter adjustment was made evaluating three kinetic models to represent respiration: 1) Power-Law Chemical kinetics (PL), 2) Simple Michaelis–Menten kinetics (MM), and 3) Michaelis–Menten with uncompetitive inhibition by CO2 (MMU). The temperature-dependence was considered in the three models by integrating an Arrhenius equation in the kinetic parameters. Afterward, a combined model was obtained to describe the fruit transpiration, coupling the moisture sorption isotherm (logistic model), and including the temperature-dependence and the most suitable respiration kinetics. It was determined that with the MMU kinetics it was possible to obtain a high goodness of fit with the experimental behaviour for the changes in the O2 and CO2 concentrations (R2adj. = 0.964 and 0.981). The MM kinetics had an acceptable fit (R2adj. = 0.940 and 0.941), while the PLK model could not explain the observed behaviour (R2adj. = 0.247 and 0.647). It was found that a logistic model appropriately described the relationship between the fruit moisture content and aw (R2adj. = 0.998). A close relationship was determined between the fruit's transpiration and its respiration rate, the change in the water activity, and the surrounding temperature and relative humidity (R2adj. = 0.998). As the temperature decreased, and as the relative humidity increased, the change in water activity and the transpiration rate decreased. Through the parameters established for each model, it is possible to predict the behaviour of processes such as respiration, transpiration, and changes in aw to configure a storage and packaging system by proposing the gas balance equations (O2, CO2, and water vapour) that allow obtaining favourable gas levels for the preservation of the fresh fruits.

Structural and Functional Characterization of NadR from Lactococcus lactis.

NadR is a bifunctional enzyme that converts nicotinamide riboside (NR) into nicotinamide mononucleotide (NMN), which is then converted into nicotinamide adenine dinucleotide (NAD). Although a crystal structure of the enzyme from the Gram-negative bacterium Haemophilus influenzae is known, structural understanding of its catalytic mechanism remains unclear. Here, we purified the NadR enzyme from Lactococcus lactis and established an assay to determine the combined activity of this bifunctional enzyme. The conversion of NR into NAD showed hyperbolic dependence on the NR concentration, but sigmoidal dependence on the ATP concentration. The apparent cooperativity for ATP may be explained because both reactions catalyzed by the bifunctional enzyme (phosphorylation of NR and adenylation of NMN) require ATP. The conversion of NMN into NAD followed simple Michaelis-Menten kinetics for NMN, but again with the sigmoidal dependence on the ATP concentration. In this case, the apparent cooperativity is unexpected since only a single ATP is used in the NMN adenylyltransferase catalyzed reaction. To determine the possible structural determinants of such cooperativity, we solved the crystal structure of NadR from L. lactis (NadRLl). Co-crystallization with NAD, NR, NMN, ATP, and AMP-PNP revealed a ‘sink’ for adenine nucleotides in a location between two domains. This sink could be a regulatory site, or it may facilitate the channeling of substrates between the two domains.

Identification of metabolites of MDR-1339, an inhibitor of β-amyloid protein aggregation, and kinetic characterization of the major metabolites in rats

We previously reported that MDR-1339, an inhibitor of β-amyloid protein aggregation, was likely to be eliminated by biotransformation in rats. The objective of this study was to determine the chemical identity of metabolites derived from this aggregate inhibitor and to characterize the kinetics of formation of these metabolites in rats. Using high performance liquid chromatography coupled with mass spectrometry with a hybrid triple quadrupole-linear ion trap, 7 metabolites and 1 potential metabolic intermediate were identified in RLM incubations containing MDR-1339. In addition to these, 3 glucuronide metabolites were detected in urine samples from rats receiving a 10 mg/kg oral dose of MDR-1339. When the kinetics of the formation of two major metabolites, M1 and M2, were analyzed assuming simple Michaelis-Menten kinetics, the Vmax and Km values were found to be 0.459 ± 0.0196 nmol/min/mg protein and 28.3 ± 3.07 μM for M1, and 0.101 ± 0.00537 nmol/min/mg protein and 14.7 ± 2.37 μM for M2, respectively. When chemically synthesized M1 and M2 were individually administered to rats intravenously at the dose of 5 mg/kg respectively, the volume of distribution and elimination clearance were determined to be 4590 ± 709 mL/kg and 68.4 ± 5.60 mL/min/kg for M1 and 15300 ± 8110 mL/kg and 98.0 ± 19.5 mL/min/kg for M2, respectively. When MDR-1339 was intravenously administered to rats at a dose of 5 mg/kg, the parent drug and M1 were readily detected for periods of up to 6 h after the administration, but M2 was observed only from 2 to 4 h. A standard moment analysis indicates that the formation clearance of M1 is 6.01 mL/min/kg, suggesting that 19.7% of the MDR-1339 dose was eliminated in rats. These observations indicate that the hepatic biotransformation of MDR-1339 results in the formation of at least 10 metabolites and that M1 is the major metabolite derived from this aggregation inhibitor in rats.

Deciphering the Biochemical Pathway and Pharmacokinetic Study of Amyloid βeta-42 with Superparamagnetic Iron Oxide Nanoparticles (SPIONs) Using Systems Biology Approach.

Alzheimer's disease (AD) pathogenesis leads to the appearance of senile plaques due to the production and deposition of the β-amyloid peptide (Aβ). Superparamagnetic iron oxide nanoparticles (SPIONs) have potential role in the detection and imaging of Aβ plaques in AD. SPIONs have shown appropriate potential in the diagnosis and treatment of AD. In the present study, the pharmacokinetics of SPIONs and its effect in the biochemical pathway of AD were analyzed using collected information. During analysis, the interaction of SPIONs with amyloid beta-42 (Aβ42), a biomarker for AD progression, has been shown. Nodes represent the entities and edges represent the relation (interactions) of one node to another node. Aβ42 and their interaction with other entities making up biochemical network are involved in AD mechanism in presence of SPION. The kinetic simulation was done to investigate pharmacokinetics of SPIONs for AD, where concentration was assigned of nanoparticles and other entities were applied as a kinetic irreversible simple Michaelis-Menten or mass action kinetics. Simulation was done in presence and absence of SPIONs to investigate pharmacokinetic effect in AD and explore the mechanism of Aβ42 in presence of SPIONs. This study may lead to better understanding, which is required to target the metabolism of Aß42 peptide, a pivotal player in this pathology.

The polycyclic aromatic hydrocarbons benzo[a]pyrene and phenanthrene inhibit intestinal lipase activity in rainbow trout (Oncorhynchus mykiss)

Elevated levels of polycyclic aromatic hydrocarbons (PAHs) are detected in aquafeeds where fish oils are (partially) replaced by vegetable oils. The highly lipophilic PAHs solubilize readily in oil droplets and micelles in the intestinal lumen that can affect enzymatic lipid digestion by altering lipase activity. We therefore investigated the effect of two PAHs, benzo[a]pyrene (BaP) and phenanthrene (PHE), on bile salt-activated lipase (BAL) activity in desalted luminal extracts of the proximal intestine of rainbow trout (Oncorhynchus mykiss) using the triacylglycerides rapeseed oil and fish oil as substrates.The hydrolysis of rapeseed oil and fish oil measured at a calculated substrate concentration of 2.2mM, increased linearly up to 30min at 15°C. Substrate dependency under initial velocity conditions was described by simple Michaelis-Menten kinetics with a Km value of 1.2mM for rapeseed and fish oil. Rapeseed oil hydrolysis was inhibited by 1nM BaP and 10nM PHE. The hydrolysis of fish oil was only inhibited by 10μM BaP. The in vitro lipase activity data were corroborated by TLC/HPLC analysis of the reaction products, showing that in the presence of BaP and PHE, 46–80% less free fatty acids (FFA) were hydrolysed from rapeseed and fish oil triacylglycerides.The presence of low concentrations of BaP and PHE decreased rapeseed oil hydrolysis by BAL whereas fish oil hydrolysis was not affected. The replacement of fish oil by rapeseed oil in aquafeeds introduces PAHs that could affect lipid digestion.

Dependence of the Enzymatic Velocity on the Substrate Dissociation Rate.

Enzymes are biological catalysts that play a fundamental role in all living systems by supporting the selectivity and the speed for almost all cellular processes. While the general principles of enzyme functioning are known, the specific details of how they work at the microscopic level are not always available. Simple Michaelis-Menten kinetics assumes that the enzyme-substrate complex has only one conformation that decays as a single exponential. As a consequence, the enzymatic velocity decreases as the dissociation (off) rate constant of the complex increases. Recently, Reuveni et al. [ Proc. Natl. Acad. Sci. USA 2014 , 111 , 4391 - 4396 ] showed that it is possible for the enzymatic velocity to increase when the off rate becomes higher, if the enzyme-substrate complex has many conformations which dissociate with the same off rate constant. This was done using formal mathematical arguments, without specifying the nature of the dynamics of the enzyme-substrate complex. In order to provide a physical basis for this unexpected result, we derive an analytical expression for the enzymatic velocity assuming that the enzyme-substrate complex has multiple states and its conformational dynamics is described by rate equations with arbitrary rate constants. By applying our formalism to a complex with two conformations, we show that the unexpected off rate dependence of the velocity can be readily understood: If one of the conformations is unproductive, the system can escape from this "trap" by dissociating, thereby giving the enzyme another chance to form the productive enzyme-substrate complex. We also demonstrate that the nonmonotonic off rate dependence of the enzymatic velocity is possible not only when all off rate constants are identical, but even when they are different. We show that for typical experimentally determined rate constants, the nonmonotonic off rate dependence can occur for micromolar substrate concentrations. Finally, we discuss the relation of this work to the problem of optimizing the flux through singly occupied membrane channels and transporters.

Purification and Biochemical Characterization of a Novel Alkaline Protease Produced by Penicillium nalgiovense

Penicillium nalgiovense PNA9 produces an extracellular protease during fermentation with characteristics of growth-associated product. Enzyme purification involved ammonium sulfate precipitation, dialysis, and ultrafiltration, resulting in 12.1-fold increase of specific activity (19.5 U/mg). The protein was isolated through a series of BN-PAGE and native PAGE runs. ESI-MS analysis confirmed the molecular mass of 45.2 kDa. N-Terminal sequencing (MGFLKLLKGSLATLAVVNAGKLLTANDGDE) revealed 93 % similarity to a Penicillium chrysogenum protease, identified as major allergen. The protease exhibits simple Michaelis-Menten kinetics and K m (1.152 mg/ml), V max (0.827 mg/ml/min), and k cat (3.2 × 10(2)) (1/s) values against azocasein show that it possesses high substrate affinity and catalytic efficiency. The protease is active within 10-45 °C, pH 4.0-10.0, and 0-3 M NaCl, while maximum activity was observed at 35 °C, pH 8.0, and 0.25 M NaCl. It is active against the muscle proteins actin and myosin and inactive against myoglobin. It is highly stable in the presence of non-ionic surfactants, hydrogen peroxide, BTNB, and EDTA. Activity was inhibited by SDS, Mn(2+) and Zn(2+), and by the serine protease inhibitor PMSF, indicating the serine protease nature of the enzyme. These properties make the novel protease a suitable candidate enzyme in meat ripening and other biotechnological applications.

Inherent Regulation of EAL Domain-catalyzed Hydrolysis of Second Messenger Cyclic di-GMP

The universal second messenger cyclic di-GMP (cdG) is involved in the regulation of a diverse range of cellular processes in bacteria. The intracellular concentration of the dinucleotide is determined by the opposing actions of diguanylate cyclases and cdG-specific phosphodiesterases (PDEs). Whereas most PDEs have accessory domains that are involved in the regulation of their activity, the regulatory mechanism of this class of enzymes has remained unclear. Here, we use biophysical and functional analyses to show that the isolated EAL domain of a PDE from Escherichia coli (YahA) is in a fast thermodynamic monomer-dimer equilibrium, and that the domain is active only in its dimeric state. Furthermore, our data indicate thermodynamic coupling between substrate binding and EAL dimerization with the dimerization affinity being increased about 100-fold upon substrate binding. Crystal structures of the YahA-EAL domain determined under various conditions (apo, Mg(2+), cdG·Ca(2+) complex) confirm structural coupling between the dimer interface and the catalytic center. The built-in regulatory properties of the EAL domain probably facilitate its modular, functional combination with the diverse repertoire of accessory domains.

L-Malate dehydrogenase activity in the reductive arm of the incomplete citric acid cycle of Nitrosomonas europaea

The autotrophic nitrifying bacterium Nitrosomonas europaea does not synthesize 2-oxoglutarate (α-ketoglutarate) dehydrogenase under aerobic conditions and so has an incomplete citric acid cycle. L-malate (S-malate) dehydrogenase (MDH) from N. europaea was predicted to show similarity to the NADP(+)-dependent enzymes from chloroplasts and was separated from the NAD(+)-dependent proteins from most other bacteria or mitochondria. MDH activity in a soluble fraction from N. europaea ATCC 19718 was measured spectrophotometrically and exhibited simple Michaelis-Menten kinetics. In the reductive direction, activity with NADH increased from pH 6.0 to 8.5 but activity with NADPH was consistently lower and decreased with pH. At pH 7.0, the K m for oxaloacetate was 20μM; the K m for NADH was 22μM but that for NADPH was at least 10 times higher. In the oxidative direction, activity with NAD(+) increased with pH but there was very little activity with NADP(+). At pH 7.0, the K m for L-malate was 5mM and the K m for NAD(+) was 24μM. The reductive activity was quite insensitive to inhibition by L-malate but the oxidative activity was very sensitive to oxaloacetate. MDH activity was not strongly activated or inhibited by glycolytic or citric acid cycle metabolites, adenine nucleotides, NaCl concentrations, or most metal ions, but increased with temperature up to about 55°C. The reductive activity was consistently 10-20 times higher than the oxidative activity. These results indicate that the L-malate dehydrogenase in N. europaea is similar to other NAD(+)-dependent MDHs (EC 1.1.1.37) but physiologically adapted for its role in a reductive biosynthetic sequence.

Cytochrome P450 1A2 forms catalytically relevant homomeric complexes

Our laboratory and others have shown that one P450 enzyme can affect the catalytic function of other P450s. The goal of this study was to determine if CYP1A2 forms functionally relevant homomeric complexes. The catalytic activity of CYP1A2 was measured in reconstituted systems containing CYP1A2 and varying levels of NADPH‐cytochrome P450 reductase (CPR). Interestingly, a sigmoidal response was observed with respect to [CPR] instead of the hyperbolic curve expected for simple Michaelis‐Menten kinetics – the sigmoidicity increased with increasing [CYP1A2]. A physical interaction between CYP1A2 molecules was verified using bioluminescence resonance energy transfer (BRET). Varying ratios of Renilla luciferase‐ and GFP2‐tagged CYP1A2 were cotransfected into HEK293T cells and energy transfer from luciferase to GFP2 was measured. Cotransfection of the tagged CYP1A2 proteins with increasing amounts of unlabeled CYP1A2 decreased the BRET response, indicating a specific physical interaction. Taken together, these results show that CYP1A2 forms CYP1A2•CYP1A2 complexes that exhibit altered catalytic activity.

Employing the metabolic "branch point effect" to generate an all-or-none, digital-like response in enzymatic outputs and enzyme-based sensors.

Here, we demonstrate a strategy to convert the graded Michaelis-Menten response typical of unregulated enzymes into a sharp, effectively all-or-none response. We do so using an approach analogous to the "branch point effect", a mechanism observed in naturally occurring metabolic networks in which two or more enzymes compete for the same substrate. As a model system, we used the enzymatic reaction of glucose oxidase (GOx) and coupled it to a second, nonsignaling reaction catalyzed by the higher affinity enzyme hexokinase (HK) such that, at low substrate concentrations, the second enzyme outcompetes the first, turning off the latter's response. Above an arbitrarily selected "threshold" substrate concentration, the nonsignaling HK enzyme saturates leading to a "sudden" activation of the first signaling GOx enzyme and a far steeper dose-response curve than that observed for simple Michaelis-Menten kinetics. Using the well-known GOx-based amperometric glucose sensor to validate our strategy, we have steepen the normally graded response of this enzymatic sensor into a discrete yes/no output similar to that of a multimeric cooperative enzyme with a Hill coefficient above 13. We have also shown that, by controlling the HK reaction we can precisely tune the threshold target concentration at which we observe the enzyme output. Finally, we demonstrate the utility of this strategy for achieving effective noise attenuation in enzyme logic gates. In addition to supporting the development of biosensors with digital-like output, we envisage that the use of all-or-none enzymatic responses will also improve our ability to engineer efficient enzyme-based catalysis reactions in synthetic biology applications.

Independence between GTPase active sites in the Escherichia coli cell division protein FtsZ

We have analyzed the substrate kinetics of the GTPase activity of FtsZ and the effects of two different GTPase inhibitors, GDP and the slowly hydrolyzable GTP analogue GMPCPP. In the absence of inhibitors the GTPase activity follows simple Michaelis–Menten kinetics, and both GDP and GMPCPP inhibited the activity in a competitive manner. These results indicate that the GTPase active sites in FtsZ filaments are independent of each other, a feature relevant to elucidate the role of GTP hydrolysis in FtsZ function and cell division. Structured summary of protein interactionsFtsZ and FtsZbind by light scattering (View interaction).

Use of Isoform-Specific UGT Metabolism to Determine and Describe Rates and Profiles of Glucuronidation of Wogonin and Oroxylin A by Human Liver and Intestinal Microsomes

Glucuronidation via UDP-glucuronosyltransferases (or UGTs) is a major metabolic pathway. The purposes of this study are to determine the UGT-isoform-specific metabolic fingerprint (or GSMF) of wogonin and oroxylin A, and to use isoform-specific metabolism rates and kinetics to determine and describe their glucuronidation behaviors in tissue microsomes. In vitro glucuronidation rates and profiles were measured using expressed UGTs and human intestinal and liver microsomes. GSMF experiments indicated that both flavonoids were metabolized mainly by UGT1As, with major contributions from UGT1A3 and UGT1A7-1A10. Isoform-specific metabolism showed that kinetic profiles obtained using expressed UGT1A3 and UGT1A7-1A10 could fit to known kinetic models. Glucuronidation of both flavonoids in human intestinal and liver microsomes followed simple Michaelis-Menten kinetics. A comparison of the kinetic parameters and profiles suggests that UGT1A9 is likely the main isoform responsible for liver metabolism. In contrast, a combination of UGT1As with a major contribution from UGT1A10 contributed to their intestinal metabolism. Correlation studies clearly showed that UGT isoform-specific metabolism could describe their metabolism rates and profiles in human liver and intestinal microsomes. GSMF and isoform-specific metabolism profiles can determine and describe glucuronidation rates and profiles in human tissue microsomes.

Novel investigation of enzymatic biodiesel reaction by isothermal calorimetry

Isothermal calorimetry (ITC) was used to investigate solvent-free enzymatic biodiesel production. The transesterification of rapeseed oil with methanol and ethanol was catalyzed by immobilized lipase Novozym 435 at 40 °C. The aim of the study was to determine reaction enthalpy for the enzymatic transesterification and to elucidate the mass transfer and energetic processes taking place. Based on the measured enthalpy and composition change in the system, the heat of reaction at 40 °C for the two systems was determined as −9.8 ± 0.9 kJ/mole biodiesel formed from rapeseed oil and methanol, and −9.3 ± 0.7 kJ/mole when rapeseed oil and ethanol was used. Simple Michaelis–Menten kinetics was not an appropriate choice for describing the kinetics of this heterogeneous system. The experiments demonstrated the possibility of investigating complex reaction mixtures using ITC. Although it is possible to determine thermodynamic properties such as reaction enthalpy and reaction rate, the difficulty in actually measuring the true non-mass-transfer-limited reaction kinetics is exposed by the high time resolution of ITC.

An evaluation of the inhibition of human butyrylcholinesterase and acetylcholinesterase by the organophosphate chlorpyrifos oxon

Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) are enzymes that belong to the superfamily of α/β-hydrolase fold proteins. While they share many characteristics, they also possess many important differences. For example, whereas they have about 54% amino acid sequence identity, the active site gorge of acetylcholinesterase is considerably smaller than that of butyrylcholinesterase. Moreover, both have been shown to display simple and complex kinetic mechanisms, depending on the particular substrate examined, the substrate concentration, and incubation conditions. In the current study, incubation of butyrylthiocholine in a concentration range of 0.005–3.0 mM, with 317 pM human butyrylcholinesterase in vitro, resulted in rates of production of thiocholine that were accurately described by simple Michaelis–Menten kinetics, with a K m of 0.10 mM. Similarly, the inhibition of butyrylcholinesterase in vitro by the organophosphate chlorpyrifos oxon was described by simple Michaelis–Menten kinetics, with a k i of 3048 nM −1 h −1, and a K D of 2.02 nM. In contrast to inhibition of butyrylcholinesterase, inhibition of human acetylcholinesterase by chlorpyrifos oxon in vitro followed concentration-dependent inhibition kinetics, with the k i increasing as the inhibitor concentration decreased. Chlorpyrifos oxon concentrations of 10 and 0.3 nM gave k is of 1.2 and 19.3 nM −1 h −1, respectively. Although the mechanism of concentration-dependent inhibition kinetics is not known, the much smaller, more restrictive active site gorge of acetylcholinesterase almost certainly plays a role. Similarly, the much larger active site gorge of butyrylcholinesterase likely contributes to its much greater reactivity towards chlorpyrifos oxon, compared to acetylcholinesterase.

Multiple Substrate Binding by Cytochrome P450 3A4: Estimation of the Number of Bound Substrate Molecules

Cytochrome P450 3A4, a major drug-metabolizing enzyme in man, is well known to show non-Michaelis-Menten steady-state kinetics for a number of substrates, indicating that more than one substrate can bind to the enzyme simultaneously, but it has proved difficult to obtain reliable estimates of exactly how many substrate molecules can bind. We have used a simple method involving studies of the effect of large inhibitors on the Hill coefficient to provide improved estimates of substrate stoichiometry from simple steady-state kinetics. Using a panel of eight inhibitors, we show that at least four molecules of the widely used CYP3A4 substrate 7-benzyloxyquinoline can bind simultaneously to the enzyme. Computational docking studies show that this is consistent with the recently reported crystal structures of the enzyme. In the case of midazolam, which shows simple Michaelis-Menten kinetics, the inhibitor effects demonstrate that two molecules must bind simultaneously, consistent with earlier evidence, whereas for diltiazem, the experiments provide no evidence for the binding of more than one molecule. The consequences of this "inhibitor-induced cooperativity" for the prediction of pharmacokinetics and drug-drug interactions are discussed.

Identification of Human Liver Cytochrome P450 Isoforms Involved in Autoinduced Metabolism of the Antiangiogenic Agent (Z)-5-[(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-propanoic Acid (TSU-68)

(Z)-5-[(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-propanoic acid (TSU-68) is a new anticancer drug that inhibits angiogenic receptor tyrosine kinases, which play a crucial role in tumor-induced vascularization. TSU-68 undergoes hepatic oxidation and glucuronidation. Incubation of TSU-68 with human liver microsomes in the presence of NADPH resulted in the formation of three major metabolites: 5-, 6-, and 7-hydroxyindolinone derivatives. The 5-, 6-, and 7-hydroxylation followed simple Michaelis-Menten kinetics with V(max)/K(m) values (an indicator of intrinsic clearance) of 13, 25, and 6 microl/min/mg, respectively. Of the 10 cDNA-expressed human cytochrome P450 isoforms examined, only CYP1A1 and CYP1A2 exhibited appreciable TSU-68 hydroxylation activity. Inhibition studies with alpha-naphthoflavone (a selective CYP1A2 inhibitor) and anti-CYP1A2 antibody also indicated the almost exclusive role of CYP1A2 in microsomal TSU-68 hydroxylation. Treatment of human hepatocytes with 10 microM TSU-68 resulted in a 28- to 140-fold increase in CYP1A1/2-mediated ethoxyresorufin O-deethylase activity. The protein levels of CYP1A2 were increased in TSU-68-treated hepatocytes, and those of CYP1A1, which were undetectable in control hepatocytes, were also increased to detectable levels in the TSU-68-treated hepatocytes. Thus, TSU-68 was shown to induce CYP1A1/2 expression, which was responsible for its hydroxylation. The observation that TSU-68 treatment resulted in a 10- to 45-fold increase in 5-, 6-, and 7-hydroxylation directly demonstrated the autoinduced hydroxylation of TSU-68. In conclusion, TSU-68 has the potential to cause induction of its own CYP1A1/2-mediated oxidative metabolism in humans. This autoinductive effect provides a clear explanation for the clinically observed decrease in TSU-68 plasma concentrations during repeated administration of the drug.

Apparent negative co-operativity and substrate inhibition in overexpressed glutamate dehydrogenase from Escherichia coli

The gene for Escherichia coli glutamate dehydrogenase (EcGDH) has been overexpressed, and a simplified purification procedure afforded greatly increased yields of c. 40 mg pure EcGDH L(-1) culture. EcGDH was unstable at a low concentration in plastic tubes, but stabilization measures allowed a robust kinetic characterization. Contrary to past reports, EcGDH deviates from Michaelis-Menten kinetics, exhibiting apparent mild negative co-operativity with both l-glutamate and NADP+, with Hill coefficients of 0.90 and 0.92, respectively. NADPH yielded simple Michaelis-Menten kinetics but both 2-oxoglutarate and NH4+ showed substrate inhibition. pH optima were 9 for oxidative deamination and 8 for reductive amination.

Kinetics of Ruminal Lipolysis of Triacylglycerol and Biohydrogenation of Long-Chain Fatty Acids: New Insights from Old Data

Previous investigations into ruminal lipolysis of triacylglycerol and ruminal biohydrogenation (BH) of unsaturated long-chain fatty acids have generally quantified these processes with either zero-order or first-order kinetics. This investigation examined if Michaelis-Menten and other nonlinear kinetics might be useful for quantifying these processes. Data from 2 previously published in vitro experiments employing rumen fluid from sheep to investigate the lipolysis of trilinolein, the BH of cis-9, cis-12 linoleic acid (LA), and the BH of fatty acids derived from the lipolysis of trilinolein were used for the development of a multi-compartmental model. The model described the lipolysis of triacylglycerol well. The model also provided a good mathematical description of the resulting production of nonesterified fatty acids, the isomerization of nonesterified LA, and subsequent production of rumenic acid (RA), vaccenic acid (VA), and stearic acid (SA). However, the model described poorly the patterns of the concentrations of LA, RA, VA, and SA after incubation of trilinolein in rumen fluid. The model is consistent with known stoichiometry and biochemistry and is parsimonious in that it employs a minimal number of parameters to describe all of the major aspects of lipolysis and BH. The first step in the lipolysis of trilinolein was described by Michaelis-Menten kinetics (Vmax=529±16mg/L per h; Km=698±41mg/L). Both subsequent lipolysis steps were approximated by a first-order (linear kinetics) rate constant (k=2.64±0.041/h). Isomerization of LA to RA was modeled by simple Michaelis-Menten kinetics (Vmax=2,421±83mg/L per h; Km=440±22mg/L). The kinetics of the BH of RA to VA was described by a Michaelis-Menten-type process involving competitive inhibition by VA (Vmax=492±6.5mg/L per h; Km=1mg/L). The final step, the BH of VA to SA, was modeled by a quasi-first-order process (k=0.533±0.021 /h), but as the concentration of VA increased, its BH appeared to be self-inhibited such that when the concentration of VA acid exceeded 517±10.4mg/L, BH was completely inhibited. The major new insights and benefits afforded by this model are 1) lipolysis and BH are described by nonlinear kinetics; 2) high concentrations of VA appear to inhibit its own BH; and 3) BH of RA appears to proceed at a much greater rate when triglyceride is present in the incubation medium. This model provides a conceptual framework for researching ruminal lipolysis and BH.

Permeability, Transport, and Metabolism of Solutes in Caco-2 Cell Monolayers: A Theoretical Study

We explored the properties of a catenary model that includes the basolateral (B), apical (A), and cellular compartments via simulations under linear and nonlinear conditions to understand the asymmetric observations arising from transporters, enzymes, and permeability in Caco-2 cells. The efflux ratio (EfR; P(app,B-->A)/P(app,A-->B)), obtained from the effective permeability from the A-->B and B-->A direction under linear conditions, was unity for passively permeable drugs whose transport does not involve transporters; the value was unaffected by cellular binding or metabolism, but increased with apical efflux. Metabolism was asymmetric, showing lesser metabolite accrual for the B-->A than A-->B direction because of inherent differences in the volumes for A and B. Moreover, the net flux (total - passive permeation) due to saturable apical efflux, absorption, or metabolism showed nonconformity to simple Michaelis-Menten kinetics against C(D,0), the loading donor concentration. EfR values differed with saturable apical efflux and metabolism (>1), as well as apical absorption (EfRs <1), but approached unity with high passive diffusive clearance (CL(d)) and increasing C(D,0) at a higher degree of saturation of the process. The J(max) (apparent V(max) estimated for the carrier system) and K(m)(') [or the K(m)('') based on a modified equation with the Hill coefficient (beta)] estimates from the Eadie-Hofstee plot revealed spurious correlations with the assigned V(max) and K(m). The sampling time, CL(d), and parameter space of K(m) and V(max) strongly influenced both the correlation and accuracy of estimates. Improved correlation was found for compounds with high CL(d). These observations showed that the catenary model is appropriate in the description of transport and metabolic data in Caco-2 cells.