Enzyme Kinetics

An important application of liquid cell Atomic Force Microscopy (AFM) is the study of enzyme structure and behaviour in organized molecular media that mimic in-vivo systems. In this study we demonstrate the use of AFM as a tool to study the kinetics of lipolytic enzyme reactions occurring at the surface of a supported lipid bilayer. In particular, the time course of the degradation of lipid bilayers by Phospholipase A 2 (PLA 2 ) and Humicola Lanuginosa Lipase (HLL) has been investigated. Contact mode imaging allows visualization of enzyme activity on the substrate with high lateral resolution. Lipid bilayers were prepared by the Langmuir-Blodgett technique and transferred to an AFM liquid cell. Following injection of the enzyme into the liquid cell, a sequence of images was acquired at regular time intervals to allow the identification of substrate structure, preferred sites of enzyme activation, and enzyme reaction rates.

A simple method to determine the in vitro catalytic turnover constant of several substrates of herpes simplex virus type 1 thymidine kinase is presented in this study. The method is based on a continuous spectroscopic enzyme-coupled assay and allows one to monitor the herpes simplex virus type 1 thymidine kinase activity in the presence of unlabeled substrates. A clear correlation between the catalytic turnover constant and the rate of decrease in absorbance over time during the assay has been demonstrated. Exploiting this correlation, this method has been used to determine rapidly and precisely the catalytic turnover constant of antiviral lead compounds not readily available in the radioactive labeled form.

Protein kinases constitute one of the largest protein families in nature. Current methods to assay their activity involve the use of radioactive ATP or very expensive reagents. In this work, we developed a highly sensitive, cost-effective and straightforward protocol to measure protein kinase activity using a microplate layout. Released ADP is converted into NAD, which is quantified by its fluorescent properties after alkaline treatment (linear range 0-10 nmol ADP). To validate our protocol, we characterized a recombinant calcium-dependent protein kinase from potato. Overall, this tool represents a critical step forward in the functional characterization of protein kinases.

The kinetic and (supra)molecular properties of the ultrasensitive behaviour of ADP-glucose pyrophosphorylase (AGPase) from Anabaena PCC 7120 (a cyanobacterium) were exhaustively studied. The response of the enzyme toward the allosteric activator 3-phosphoglycerate (3PGA) occurs with ultrasensitivity as a consequence of the cross-talk with the inhibitor P i . Molecular ' crowding ' renders AGPase more sensitive to the interplay between the allosteric regulators and, consequently, enhances the ultrasensitive response. In crowded media, and when orthophosphate is present, the activation kinetics of the enzyme with 3PGA proceed with increased co-operativity and reduced affinity toward the activator. Under conditions of ultrasensitivity, the enzyme's maximal activation takes place in a narrow range of 3PGA concentrations. Moreover, saturation kinetics of the enzyme with respect to its substrates, glucose 1-phosphate and ATP, were different at low or high 3PGA levels in crowded media. Only under the latter conditions did AGPase exhibit Abbreviations used : AGPase, ADP-glucose pyrophosphorylase ; 3PGA, 3-phosphoglycerate ; PEG, poly(ethylene glycol) ; DTT, dithiothreitol. 1 To whom correspondence should be addressed (e-mail iglesias!criba.edu.ar).

The effect of the low-molecular-mass natural reagents in high concentrations is important for investigating enzymatic reactions in near "in vivo" conditions and for optimisation of biotechnology processes. A model system, including p-nitrophenyl acetate as substrate and ®-chymotrypsin as proteolytic enzyme, has been used to study the effect of high concentrations of sucrose, both in uencing the viscosity of the reaction medium and acting as a nucleophilic effector (activator) on the enzymatic reaction. A kinetic scheme at high concentrations of nucleophilic effectors (sucrose) has been proposed.

In this paper, we present the first detailed analysis of the modes of action of three purified, thermostable endo-␤-d-glucanases (EG V-VII) against a range of soluble ␤-linked glucans. Studies indicated that EG V-VII, purified to homogeneity from a new source, the thermophilic fungus Talaromyces emersonii, are strict ␤-glucanases that exhibit maximum activity against mixed-link 1,3;1,4-␤-d-glucans. Time-course hydrolysis studies of 1,4-␤-d-glucan (carboxymethylcellulose; CMC), 1,3;1,4-␤-d-glucan from barley (BBG) and lichenan confirmed the endo-acting nature of EG V-VII and verified preference for 1,3;1,4-␤-d-glucan substrates. The results suggest that EG VI and EG VII belong to EC 3.2.1.6, as both enzymes also exhibit activity against 1,3-␤-glucan (laminaran), in contrast to EG V. Although cellobiose, cellotriose and glucose were the main glucooligosaccharide products released, the range and relative amount of each product was dependent on the particular enzyme, substrate and reaction time. Kinetic constants (K m , V max , k cat and k cat /K m ) determined for EG V-VII with BBG as substrate yielded similar K m and V max values for EG V and EG VI. EG VII exhibited highest affinity for BBG (K m value of 9.1 mg ml -1 ) and the highest catalytic efficiency (k cat /K m of 12.63 s -1 mg -1 ml).

The regulatory mechanism of substrate activation in yeast pyruvate decarboxylase is triggered by the interaction of pyruvic acid with C221 located on the L domain at s 20 A î from the thiamin diphosphate (ThDP). To trace the putative information transfer pathway, substitutions were made at H92 on the K domain, across the domain divide from C221, at E91, next to H92 and hydrogen bonded to W412, the latter being intimately involved in the coenzyme binding locus. Additional substitutions were made at D28, E51, H114, H115, I415 and E477, all near the active center. The pH-dependent steady-state kinetic parameters, including the Hill coefficient, provide useful insight to this effort. In addition to C221, the residues H92, E91, E51 and H114 and H115 together appear to have a critical impact on the Hill coefficient, providing a pathway for information transfer. To study the activation by ThDP.Mg(II), variants at G231 (of the conserved GDG triplet) and at N258 and C259 (all three being part of the putative ThDP fold) of the E1 component of the Escherichia coli pyruvate dehydrogenase multienzyme complex were studied. Kinetic and spectroscopic evidence suggests that the Mg(II) ligands are very important to activation of the enzymes by cofactors.

A series of substituted aryl -glycosides derived from 3-O--cellobiosyl-d-glucopyranose with dierent phenol-leaving group abilities as measured by the pK a of the free phenol group upon enzymatic hydrolysis has been synthesised. Aryl -glycosides with a pK a of the free phenol leaving group>5 were prepared by phase-transfer glycosidation of the per-O-acetylated -glycosyl bromide with the corresponding phenol, whereas the 2,4-dinitrophenyl -glycoside was obtained by condensation of 1-¯uoro-2,4-dinitrobenzene with the partially acetylated trisaccharide followed by acid de-O-acetylation. The aryl -glycosides have been used for reactivity studies of the wild-type Bacillus licheniformis 1,3-1,4--d-glucan 4-glucanohydrolase. The Hammett plot log k t versus pK a is biphasic with an upward curvature at low pK a values suggesting a change in transition-state structure depending on the aglycon.

In this study, we show that oxygen regulates nitric oxide (NO) levels through effects on NO synthase (NOS) enzyme kinetics. Initially, NO synthesis in the static lung was measured in bronchiolar gases during an expiratory breath-hold in normal individuals. NO accumulated exponentially to a plateau, indicating balance between NO production and consumption in the lung. Detection of NO 2 Ϫ , NO 3 Ϫ , and S-nitrosothiols in lung epithelial lining fluids confirmed NO consumption by chemical reactions in the lung. Interestingly, alveolar gas NO (estimated from bronchiolar gases at endexpiration) was near zero, suggesting NO in exhaled gases is not derived from circulatory/systemic sources. Dynamic NO levels during tidal breathing in different airway regions (mouth, trachea, bronchus, and bronchiole) were similar. However, in individuals breathing varying levels of inspired oxygen, dynamic NO levels were notably dependent on O 2 concentration in the hypoxic range ( K m O 2 190 M). Purified NOS type II enzyme activity in vitro was similarly dependent on molecular oxygen levels ( K m O 2 135 M), revealing a means by which oxygen concentration affects NO levels in vivo. Based upon these results, we propose that NOS II is a mediator of the vascular response to oxygen in the lung, because its K m O 2 allows generation of NO in proportion to the inspired oxygen concentration throughout the physiologic range. (

Humicolagriseavar.thermoideais an aerobic and thermophilic fungus that secretes the GH11 xylanase HXYN2 in the presence of sugarcane bagasse. In this study, HXYN2 was expressed inPichiapastorisand characterized biochemically and structurally in the presence of beechwood xylan substrate and ferulic acid (FA). HXYN2 is a thermally stable protein, as indicated by circular dichroism, with greater activity in the range of 40–50 °C and pH 5.0–9.0, with optimal temperature and pH of 50 °C and 6.0, respectively. FA resulted in a 75% increase in enzyme activity and a 2.5-fold increase in catalytic velocity, catalytic efficiency, and catalytic rate constant (kcat), with no alteration in enzyme affinity for the substrate. Fluorescence quenching indicated that FA forms a complex with HXYN2 interacting with solvent-exposed tryptophan residues. The binding constants ranged from moderate (pH 7.0 and 9.0) to strong (pH 4.0) affinity. Isothermal titration calorimetry, structural models and molecular...

Mitochondrial uncoupling proteins are a proton transporter family involved in regulation mitochondrial superoxide and ATP production. Uncoupling proteins are expressed by rat spiral ganglion and vestibular ganglion cells [Hear Res 196 (2004) 39]. This study tests the hypothesis that uncoupling protein expression is up-regulated in response to the reactive oxygen species challenge imposed by kanamycin and antioxidant (2,3-dihydroxybenzoate) treatment in mice. In control C57BL/6, CBA/J and BALB/c mice, mRNA for uncoupling protein 1, uncoupling protein 2, uncoupling protein 3, Slc25a27 (uncoupling protein 4) and Slc25a14 (uncoupling protein 5/BMCP1) was expressed in the spiral and vestibular ganglia. After kanamycin-treatment (700 mg/kg twice daily for 14 days s.c.), uncoupling protein 2 and uncoupling protein 3 mRNA expression increased significantly in spiral and vestibular ganglia and kidney, but was unaffected in cerebral cortex. Significant Slc25a27 (uncoupling protein 4) mRNA up-regulation was also observed in spiral and vestibular ganglia, but not in kidney or cerebral cortex. These effects were blocked by simultaneous administration of kanamycin and 2,3-dihydroxybenzoate (300 mg/kg twice daily for 14 days s.c.). Western immunoblotting and immunohistochemistry confirmed the uncoupling protein 2 and uncoupling protein 3 up-regulation in inner ear. Finally, 2,3-dihydroxybenzoate treatment alone produced an upregulation of uncoupling protein 1 mRNA in the spiral ganglion, vestibular ganglion and cerebral cortex, but not the kidney. Uncoupling protein 2 and uncoupling protein 3 upregulation in the kidney and inner ear ganglia likely reflects their general role as a feedback pathway to reduce mitochondrial superoxide generation. Slc25a27 (uncoupling protein 4) upregulation in the inner ear ganglia, by contrast, is likely to be a secondary response to kanamycin-induced hair cell death. We propose that increased uncoupling protein 2, uncoupling protein 3 and Slc25a27 expression has several neuroprotective effects via reduction in mitochondrial superoxide generation and local thermogenesis, including: (1) reducing mean ROS load to prevent apoptosis, (2) increasing signal-to-noise characteristics of intracellular ROS signaling pathways (e.g. lipoxygenases, growth factor and transcription factors), (3) heatrelated alteration of enzyme kinetics and (4) promotion of cell depolarization (activation of heat-gated ion channels).

In this study, we show that oxygen regulates nitric oxide (NO) levels through effects on NO synthase (NOS) enzyme kinetics. Initially, NO synthesis in the static lung was measured in bronchiolar gases during an expiratory breath-hold in normal individuals. NO accumulated exponentially to a plateau, indicating balance between NO production and consumption in the lung. Detection of NO 2 Ϫ , NO 3 Ϫ , and S-nitrosothiols in lung epithelial lining fluids confirmed NO consumption by chemical reactions in the lung. Interestingly, alveolar gas NO (estimated from bronchiolar gases at endexpiration) was near zero, suggesting NO in exhaled gases is not derived from circulatory/systemic sources. Dynamic NO levels during tidal breathing in different airway regions (mouth, trachea, bronchus, and bronchiole) were similar. However, in individuals breathing varying levels of inspired oxygen, dynamic NO levels were notably dependent on O 2 concentration in the hypoxic range ( K m O 2 190 M). Purified NOS type II enzyme activity in vitro was similarly dependent on molecular oxygen levels ( K m O 2 135 M), revealing a means by which oxygen concentration affects NO levels in vivo. Based upon these results, we propose that NOS II is a mediator of the vascular response to oxygen in the lung, because its K m O 2 allows generation of NO in proportion to the inspired oxygen concentration throughout the physiologic range. (

A steady-state kinetic study of bovine serum amine oxidase activity was performed, over a wide range of pH values (5.4–10.2) and ionic strength (10–200mM), using various (physiological and analogue) substrates as specific probes of the active-site binding region. Relatively small changes in kcat values (approx. one order of magnitude) accompanied by marked changes in Km and kcat/Km values (approx. six orders of magnitude) were observed. This behaviour was correlated with the presence of positively charged groups or apolar chains in the substrates. In particular, it was found that the docking of the physiological polyamines, i.e. spermidine and spermine, appears to be modulated by three amino acid residues of the active site, which we have named L-H+, G-H+ and IH+, characterized by pKa values of 6.2±0.2 [Di Paolo, Scarpa, Corazza, Stevanato and Rigo (2002) Biophys. J. 83, 2231–2239], 8.2±0.3 and 7.8±0.4 respectively. The electrostatic interaction between the protonated substrates and...

Pyrosequencing is a DNA sequencing technique based on sequencing-by-synthesis enabling rapid real-time sequence determination. This technique employs four enzymatic reactions in a single tube to monitor DNA synthesis. Nucleotides are added iteratively to the reaction and in case of incorporation, pyrophosphate (PPi) is released. PPi triggers a series of reactions resulting in production of light, which is proportional to the amount of DNA and number of incorporated nucleotides. Generated light is detected and recorded by a detector system in the form of a peak signal, which reflects the activity of all four enzymes in the reaction. We have developed simulations to model the kinetics of the enzymes. These simulations provide a full model for the Pyrosequencing four-enzyme system, based on which the peak height and shape can be predicted depending on the concentrations of enzymes and substrates. Simulation results are shown to be compatible with experimental data. Based on these simulations, the rate-limiting steps in the chain can be determined, and K M and k cat of all four enzymes in Pyrosequencing can be calculated.

In this paper the response of an amperometric biosensor at mixed enzyme kinetics and diffusion limitation in the cases of both enzyme entrapped within the conducting polymer film and in bulk solution is discussed. The model is based on diffusion equations containing a non-linear term related to Michaelis-Menten kinetics of the enzymatic reaction processes. New Homotopy perturbation method is employed to solve the system of coupled non-linear diffusion equations for the non-steady-state condition. Simple and accurate general analytical expressions of concentration of substrate, hydrogen peroxide and flux are derived for all possible values of parameters. A numerical simulation was carried out using Matlab/Scilab program. The analytical results are compared with the numerical results and found to be in good agreement. The results obtained in this work are valid for the entire solution domain

We have used ribonuclease T1 and its chemically modified derivatives as substrates, and trypsin as proteinase, to investigate the kinetics of proteolysis of a specific pep- tide bond in the folded and unfolded conformations of a protein. Steady-state kinetic studies showed that Km = 0.27mM and Kcat. = 2.45s-s for the tryptic hydrolysis of the Arg(77)-Val(78) peptide bond in unfolded ribonuclease T1. This Km is somewhat lower than, and the kcat. value similar to, values found for the tryptic hydrolysis of comparable bonds in small peptides. Our data for the initial velocity of hydrolysis of the Arg(7-7)-Val(78) bond in a solution of the folded protein indicate that the bond is at least 1700 times less rapidly hydrolysed in the folded than in the unfolded conform- ation of ribonuclease T1, and do not exclude the possibility that the bond is com- pletely resistant to hydrolysis in the folded protein. Peptide-bond cleavage plays an essential role in many important biological processes, including the post-translational processing steps of protein syn- thesis, the generation of polypeptide hormones, assembly of intracellular organelles, blood clotting, intracellular protein degradation, tumour invasion and alimentary digestion. It is therefore desirable that we understand the kinetics of peptide-bond cleavage by proteolytic enzymes. Most of our knowledge of the action of proteinases is based on kineties studies of the hydrolysis of small peptides, so that much is known of the influence of covalent structure on the kinetics of proteolysis, but there is little information on the influence of conformation, since most small peptides do not form stable folded conformations in aqueous solution (Blundell & Wood, 1982). It is well established that most proteins are ren- dered much more susceptible to proteolysis by denaturation. The significance of this has been much debated since the seminal work of Lin- derstr0m-Lang's laboratory in the late 1940s Abbreviations used: the abbreviations used for amino acid derivatives and N-terminal groups are based on the standard conventions [Biochem. J. (1972) 126, 773-780]. Ribonuclease T, derivatives: Ac, N-acetyl; Cm, S- carboxymethyl; Suc, N-2-carboxypropionyl.

catalyzed conjugation with glucuronic acid to form morphine 3and 6-glucuronides (M3G and M6G, respectively). It has been proposed that UGT2B7 is the major enzyme involved in these reactions, but there is evidence to suggest that other isoforms also catalyze morphine glucuronidation in man. Thus, we have characterized the selectivity and kinetics of M3G and M6G formation by recombinant human UGTs. UGT 1A1, 1A3, 1A6, 1A8, 1A9, 1A10, and 2B7 all catalyzed M3G formation, but only UGT2B7 formed M6G. The kinetics of M3G formation by the UGT1A family isoforms was consistent with a single enzyme Michaelis-Menten model, with apparent K m values ranging from 2.6 to 37.4 mM. In contrast, M3G and M6G formation by UGT2B7 exhibited atypical kinetics. The atypical kinetics may be described by a model with high-and low-affinity K m values (0.42 and 8.3 mM for M3G, and 0.97 and 7.4 mM for M6G) from fitting to a biphasic Michaelis-Menten model. However, a multisite model with an interaction between two identical binding sites in a negative cooperative manner provides a more realistic approach to modeling these data. According to this model, the respective binding affinities (K s ) for M3G and M6G were 1.76 and 1.41 mM, respectively. These data suggest that M6G formation may be used as a selective probe for UGT2B7 activity, and morphine glucuronidation by UGT2B7 appears to involve the simultaneous binding of two substrate molecules, highlighting the need for careful analysis of morphine glucuronidation kinetics in vitro. Chemicals. Morphine hemisulfate was a gift from Faulding Pharmaceuticals (Adelaide, Australia). M6G was purchased from Ultrafine Chemicals (Manchester, UK), and M3G, UDP-glucuronic acid (UDPGA), 4-methylum-

The Michaelis-Menten model, and the existence of a single active site for the interaction of substrate with drug metabolizing enzyme, adequately describes a substantial number of in vitro metabolite kinetic data sets for both clearance and inhibition determination. However, in an increasing number of cases (involving most notably, but not exclusively, CYP3A4), atypical kinetic features are observed, e.g., auto-and heteroactivation; partial, cooperative, and substrate inhibition; concentration-dependent effector responses (activation/inhibition); limited substrate substitution and inhibitory reciprocity necessitating sub-group classification. The phenomena listed above cannot be readily interpreted using single active site models and the literature indicates that three types of approaches have been adopted. First the Ônaı ¨veÕ approach of using the Michaelis-Menten model regardless of the kinetic behaviour, second the ÔempiricalÕ approach (e.g., employing the Hill or uncompetitive inhibition equations to model homotropic phenomena of sigmoidicity and substrate inhibition, respectively) and finally, the ÔmechanisticÕ approach. The later includes multisite kinetic models derived using the same rapid equilibrium/steady-state assumptions as the single-site model. These models indicate that 2 or 3 binding sites exist for a given CYP3A4 substrate and/or effector. Multisite kinetic models share common features, depending on the substrate kinetics and the nature of the effector response observed in vitro, which allow a generic model to be proposed. Thus although more complex than the other two approaches, they show more utility and can be comprehensively applied in relatively simple versions that can be readily generated from generic model. Multisite kinetic features, observed in isolated hepatocytes as well as in microsomes from hepatic tissue and heterologous expression systems, may be evident in substrate depletion-time profiles as well as in metabolite formation rates. Failure to adequately account for multisite kinetic phenomena will compromise any attempts to predict human drug clearance and drug-drug interaction potential from in vitro data.

Prokaryotic and eukaryotic Na + /Ca 2+ exchangers (NCX) control Ca 2+ homeostasis. NCX orthologs exhibit up to 10 4 -fold differences in their turnover rates (k cat ), whereas the ratios between the cytosolic (cyt) and extracellular (ext) K m values (K int = K m Cyt /K m Ext ) are highly asymmetric and alike (K int ≤ 0.1) among NCXs. The structural determinants controlling a huge divergence in k cat at comparable K int remain unclear, although 11 (out of 12) ion-coordinating residues are highly conserved among NCXs. The crystal structure of the archaeal NCX (NCX_Mj) was explored for testing the mutational effects of pore-allied and loop residues on k cat and K int . Among 55 tested residues, 26 mutations affect either k cat or K int , where two major groups can be distinguished. The first group of mutations (14 residues) affect k cat rather than K int . The majority of these residues (10 out of 14) are located within the extracellular vestibule near the pore center. The second group of mutations (12 residues) affect K int rather than k cat , whereas the majority of residues (9 out 12) are randomly dispersed within the extracellular vestibule. In conjunction with computational modeling-simulations and hydrogen-deuterium exchange mass-spectrometry (HDX-MS), the present mutational analysis highlights structural elements that differentially govern the intrinsic asymmetry and transport rates. The key residues, located at specific segments, can affect the characteristic features of local backbone dynamics and thus, the conformational flexibility of ion-transporting helices contributing to critical conformational transitions. The underlying mechanisms might have a physiological relevance for matching the response modes of NCX variants to cell-specific Ca 2+ and Na + signaling.

Prokaryotic and eukaryotic Na + /Ca 2+ exchangers (NCX) control Ca 2+ homeostasis. NCX orthologs exhibit up to 10 4 -fold differences in their turnover rates (k cat ), whereas the ratios between the cytosolic (cyt) and extracellular (ext) K m values (K int = K m Cyt /K m Ext ) are highly asymmetric and alike (K int ≤ 0.1) among NCXs. The structural determinants controlling a huge divergence in k cat at comparable K int remain unclear, although 11 (out of 12) ion-coordinating residues are highly conserved among NCXs. The crystal structure of the archaeal NCX (NCX_Mj) was explored for testing the mutational effects of pore-allied and loop residues on k cat and K int . Among 55 tested residues, 26 mutations affect either k cat or K int , where two major groups can be distinguished. The first group of mutations (14 residues) affect k cat rather than K int . The majority of these residues (10 out of 14) are located within the extracellular vestibule near the pore center. The second group of mutations (12 residues) affect K int rather than k cat , whereas the majority of residues (9 out 12) are randomly dispersed within the extracellular vestibule. In conjunction with computational modeling-simulations and hydrogen-deuterium exchange mass-spectrometry (HDX-MS), the present mutational analysis highlights structural elements that differentially govern the intrinsic asymmetry and transport rates. The key residues, located at specific segments, can affect the characteristic features of local backbone dynamics and thus, the conformational flexibility of ion-transporting helices contributing to critical conformational transitions. The underlying mechanisms might have a physiological relevance for matching the response modes of NCX variants to cell-specific Ca 2+ and Na + signaling.

Antibacterial activity of aloe vera essential oil extract (0.8gm/100 µl) was investigated in multi-drug resistance (MDR) Staphylococcus aureus specimen isolated from patients with skin infection in Baghdad. Aloe vera essential oil extract showed obvious antistaphylococcal activity probably due to the presence of Anthraquinones components of aloe vera which include (aloin 2.3%, isobarbaloin 36.1%, anthranol 42.6%, aloectic acid 16.8%,and aloe emodin 2.1%). Fast liquid chromatography was used to separate these components. Antibiotics combinations revealed that Aloe vera essential oil with Clindamycin against MDR isolates of S. aureus showed a synergistic effect when used as 1/4 MIC for each antimicrobial. In vivo study was executed to determine antibacterial activity of these compounds by induction of skin infection with Staphylococcus aureus in mice and the treatment begun after 4hrs later and continue to seven days then skin biopsy was taken and sent for histopathological examinatio...

Resistance to some (lipophilic) antifolates has been associated with P-glycoprotein (P-gp)mediated multidrug resistance (MDR). A possible relationship with non-P-gp MDR has not been established. We studied resistance to antifolates in SW-1573 human lung carcinoma cells, a P-gp overexpressing variant SW-1573/2R160 and a multidrug resistance protein (MRP) overexpressing variant SW-1573/2R120. In this study, thymidylate synthase (TS) inhibitors with different properties concerning the efficiency of membrane transport and the efficiency of polyglutamylation were tested for cross-resistance in SW-1573/2R120 and SW-1573/2R160 cells. Growth inhibition patterns in this cell line panel were measured by the Sulforhodamine B (SRB) assay. Resistance factors for TS inhibitors were: 2.4 and 0.4 for 5-fluorouracil (5FU), 18.8 and 8.8 for ZD1694, 17 and 0.7 for AG337, and 40 and 8.3 for BW1843U89 in SW-1573/2R160 and SW-1573/2Rl20, respectively. This study showed changes in the TS enzyme kinetics during the induction of doxorubicin resistance in both SW-1573 variants, resulting in 2-fold lower K, values for 2'-deoxyuridine-5'-monophosphate (dUMP) in both resistant variants compared to the parental cell line. TS activity, TS protein induction and TS mRNA expression all had 2-fold

BACKGROUND: The present work aims to gain further insight on the use of Fusarium solani pisi cutinase from a Saccharomyces cerevisiae strain for the synthesis of short chain ethyl esters, a group of important fruit flavor compounds, in a non-conventional environment. Synthesis is promoted by cutinase in organic media, in particular, in iso-octane, an organic solvent recognized as a safe ingredient in food and beverage industrial processes. RESULTS: The effect of solvent and substrate components of the reaction mixture on the enzyme stability was measured separately. Focus was given to the effect of reaction medium on the operational stability of cutinase and on the esterification yield after 24h. The feasibility of the operation in fed batch mode was successfully evaluated. The effect of the addition of substrate in consecutive pulses in the activity and stability of the biocatalyst was again assessed. CONCLUSIONS: The bioconversion system used in this work allowed for the sustained production of short chain alkyl esters for more than 45 days, which is suggestive of the stability of cutinase in the organic environment evaluated.

UDP-glucose (UDPG) pyrophosphorylase (UGPase) produces UDPG for sucrose and polysaccharide synthesis and glycosylation reactions. In this study, several barley UGPase mutants were produced, either single amino acid mutants or involving deletions of Nand C-terminal domains (Ncut and Ccut mutants, respectively) and of active site region ("NB loop"). The Del-NB mutant yielded no activity, whereas Ncut deletions and most of Ccut mutants, including short deletions at the so called "I-loop" region of C-terminal domain, as well as a single K260A mutant resulted in very low activity. For wt and the mutants, kinetics with UDPG were linear on reciprocal plots, whereas PPi at concentrations above 1 mM exerted strong substrate inhibition. Both K260A and most of the Ccut mutants had very high K m with PPi (up to 33 mM), whereas Ncut deletions had greatly increased K m with UDPG (up to 57 mM). Surprisingly, an 8 amino acid deletion from end of the Cterminus resulted in an enzyme (Ccut-8 mutant) with 44% higher activity when compared to wt, but with similar K m values. Whereas Ccut-8 existed solely as a monomer, other deletion mutants had a more oligomerized status, e.g. Ncut mutants existing primarily as dimers. Overall, the data confirmed the essential role of NB loop in catalysis, but also pointed out to the role of both Nand C-termini for activity, substrate binding and oligomerization. The importance of oligomerization status for enzymatic activity of UGPase is discussed.

La postcosecha del banano se acompaña de múltiples reacciones enzimáticas, causantes de la degradación de almidones y clorofila, de la síntesis de azúcares y carotenos, y de los cambios en la acidez, ablandamiento de tejidos y pardeamiento enzimático. Este pardeamiento es causado por la acción de la Polifenol Oxidasa (PFO) sobre los fenoles contenidos en el fruto y es considerado un cambio indeseable por el consumidor. Con el objeto de determinar el momento de mínima actividad enzimática para lograr su óptimo procesamiento, se caracterizó la variedad Gros Michel en diferentes estados de maduración, en cuanto a su color, textura, actividad de agua, humedad y grados Brix, y luego se midió la cinética enzimática, aprovechando la formación del color café durante el pardeamiento, empleando un espectrofotómetro de UV-visible a 420nm. También se cuantificó la cantidad de proteína (enzima) mediante el método de Bradford (lectura a 595nm). Una vez identificadas las cinéticas de cada estado de maduración, se hizo una comparación entre cada uno de los grupos mediante la técnica estadística de Análisis de Varianza con Medidas Repetidas en el tiempo, que mostró una marcada diferencia, estadísticamente significativa, entre los bananos de menor y mayor estado de maduración en forma decreciente, y se encontró el punto mínimo de actividad de la enzima en los estados más avanzados de maduración.

Chronic heart failure is one of the most frequent causes of death in humans. Knockout of type 5 adenylyl cyclase (AC) in mice causes longevity and protection from cardiomyopathy, and an AC5 inhibitor reduces ␤-adrenoceptor-stimulated Ca 2ϩ inward currents in isolated mouse cardiomyocytes. These data indicate that selective AC5 inhibitors may be beneficial in chronic heart failure. Therefore, we characterized AC in mouse heart membranes. Real-time polymerase chain reaction and immunoblot analysis suggested that AC5 is an important heart AC isoform. Enzyme kinetics of heart AC and recombinant AC5 in the presence of Mg 2ϩ were similar. Moreover, the inhibitory profile of eight 2Ј(3Ј)-O-(N-methylanthraniloyl) (MANT)-nucleoside 5Ј-([␥-thio])triphosphates on mouse heart in the presence of Mg 2ϩ was almost identical to that of AC5. MANT-ITP was the most potent inhibitor of heart AC and recombinant AC5, with K i values in the 15 to 25 nM range in the presence of Mg 2ϩ and in the 1 to 5 nM range in the presence of Mn 2ϩ . However, in the presence of Mn 2ϩ , we also noted differences between mouse heart AC and AC5 with respect to enzyme kinetics and forskolin analog effects. In conclusion, with regard to expression and kinetics and inhibition by MANT-nucleotides in the presence of Mg 2ϩ , AC5 is an important AC isoform in heart, with MANT-ITP being an excellent starting point for the design of AC5-selective inhibitors. Unfortunately, a limitation of our study is the fact that immunologically and biochemically, AC5 and AC6 are quite similar, although they have different roles in heart. Moreover, lack of antibody specificity and Mn 2ϩ masking AC5 effects were problems.

The common marmoset Callithrix jacchus encodes two glutathione transferase (GST) enzymes with ketosteroid double-bond isomerase activity. The most active enzyme is CjaGST A3-3 showing a specific activity with 5androsten-3,17-dione (Δ 5 -AD) of 62.1 ± 1.8 μmol min -1 mg -1 , and a k cat value of 261 ± 49 s -1 . The second ketosteroid isomerase CjaGST A1-1 has a 30-fold lower specific activity with Δ 5 -AD and a 37-fold lower k cat value. Thus, the marmoset CjaGST A3-3 would be the main contributor to the biosynthesis of the steroid hormones testosterone and progesterone, like the human ortholog HsaGST A3-3. Two residues differ in the H-site of the 91.4% sequence identical CjaGST A1-1 and CjaGST A3-3, and modeling of the structures suggests that the bulky phenyl ring of Phe111 in CjaGST A1-1 causes steric hindrance in the binding of the steroid substrate. Tributyltin acetate (IC 50 =0.16 ± 0.004 μM) and ethacrynic acid (IC 50 =3.3 ± 0.2 μM) were found to be potent inhibitors of CjaGST A3-3, as previously demonstrated with the human and equine orthologs.

Glutathione transferases (GSTs; EC. 2.5.1.18) are a large family of multifunctional enzymes that play crucial roles in the metabolism and inactivation of a broad range of xenobiotic compounds. In the present work, we report the kinetic and structural characterization of the isoenzyme GSTM1-1 from Camelus dromedarius (CdGSTM1-1). The CdGSTM1-1 was expressed in E. coli BL21 (DE3) and was purified by affinity chromatography. Kinetics analysis showed that the enzyme displays a relative narrow substrate specificity and restricted ability to bind xenobiotic compounds. The crystal structures of CdGSTM1-1 were determined by X-ray crystallography in complex with the substrate (GSH) or the reaction product (S-p-nitrobenzyl-GSH), providing snapshots of the induced-fit catalytic mechanism. The thermodynamic stability of CdGSTM1-1 was investigated using differential scanning fluorimetry (DSF) in the absence and in presence of GSH and S-p-nitrobenzyl-GSH and revealed that the enzyme's structure is significantly stabilized by its ligands. The results of the present study advance the understanding of camelid GST detoxification mechanisms and their contribution to abiotic stress adaptation in harsh desert conditions.

The isoenzyme glutathione S-transferase (GST) I from maize (Zea mays) was cloned and expressed in Escherichia coli, and its catalytic mechanism was investigated by site-directed mutagenesis and dynamic studies. The results showed that the enzyme promotes proton dissociation from the GSH thiol and creates a thiolate anion with high nucleophilic reactivity by lowering the pK a of the thiol from 8.7 to 6.2. Steady-state kinetics fit well to a rapid equilibrium, random sequential Bi Bi mechanism, with intrasubunit modulation between the GSH binding site (G-site) and the electrophile binding site (H-site). The rate-limiting step of the reaction is viscosity-dependent, and thermodynamic data suggest that product release is rate-limiting. Five residues of GST I (Ser"", His%!, Lys%", Gln&$ and Ser'(), which are located in the Gsite, were individually replaced with alanine and their structural and functional roles in the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction were investigated. On the basis of steadystate kinetics, difference spectroscopy and limited proteolysis studies it is concluded that these residues : (1) contribute to the affinity of the G-site for GSH, as they are involved in side-chain interaction with GSH ; (2) influence GSH thiol ionization, and thus its reactivity ; (3) participate in k cat regulation by affecting the rate-limiting step of the reaction ; and (4) in the cases of His%!,

The amidase activity of human a-thrombin has been studied at steady state in the pH range 6-10, as a function of NaCl concentration from 1 mM to 1 M and temperature from 10 to 40 OC. The Michaelis-Menten constant, K,, shows a bell-shaped dependence over this pH range with a minimum around pH 7.5 in the presence of 0.1 M NaCl at 25 OC. The catalytic constant, kat, also has a bell-shaped pH dependence with multiple inflection points that are more evident at low NaCl concentrations and a maximum around pH 8.2 in the presence of 0.1 M NaCl at 25 OC. A detailed analysis of the results in terms of a general linkage scheme has allowed a thorough characterization of the linkage between proton and substrate binding and its dependence on NaCl concentration, as well as the relevant entropic and enthalpic contributions to binding and catalytic events. Formulation of detailed partition functions for each enzyme intermediate involved in the catalytic cycle suggests that (at least) three groups are responsible for the control of thrombin amidase activity as a function of pH. One group is to be identified with the active site His, due to its pK values in the free enzyme and the adduct and its enthalpy of ionization. The effect of NaCl concentration on amidase activity seems to be extremely specific. Comparative steady-state measurements carried out in the presence of NaCl, NaBr, NaI, KC1, and MgClz show that human a-thrombin is capable of discriminating among different cations and anions. This suggests that small ions participate as allosteric effectors in the regulation of thrombin activity. The linkage with NaCl is strongly pH dependent and increases with decreasing pH. The present results provide information on the basic aspects of human a-thrombin activity and regulation and enable a rigorous thermodynamic approach to other important regulatory interactions in human a-thrombin and its structurally perturbed derivatives.

Triosephosphate isomerase (TIM) is often described as a fully evolved housekeeping enzyme with near-maximal possible reaction rate. The assumption that an enzyme is perfectly evolved has not been easy to confirm or refute. In this paper, we use maximization of entropy production within known constraints to examine this assumption by calculating steady-state cyclic flux, corresponding entropy production, and catalytic activity in a reversible four-state scheme of TIM functional states. The maximal entropy production (MaxEP) requirement for any of the first three transitions between TIM functional states leads to decreased total entropy production. Only the MaxEP requirement for the product (R-glyceraldehyde-3-phosphate) release step led to a 30% increase in enzyme activity, specificity constant k cat /K M , and overall entropy production. The product release step, due to the TIM molecular machine working in the physiological direction of glycolysis, has not been identified before as the ratelimiting step by using irreversible thermodynamics. Together with structural studies, our results open the possibility for finding amino acid substitutions leading to an increased frequency of loop six opening and product release.

Triosephosphate isomerase (TIM) is often described as a fully evolved housekeeping enzyme with near-maximal possible reaction rate. The assumption that an enzyme is perfectly evolved has not been easy to confirm or refute. In this paper, we use maximization of entropy production within known constraints to examine this assumption by calculating steady-state cyclic flux, corresponding entropy production, and catalytic activity in a reversible four-state scheme of TIM functional states. The maximal entropy production (MaxEP) requirement for any of the first three transitions between TIM functional states leads to decreased total entropy production. Only the MaxEP requirement for the product (R-glyceraldehyde-3-phosphate) release step led to a 30% increase in enzyme activity, specificity constant k cat /K M , and overall entropy production. The product release step, due to the TIM molecular machine working in the physiological direction of glycolysis, has not been identified before as the ratelimiting step by using irreversible thermodynamics. Together with structural studies, our results open the possibility for finding amino acid substitutions leading to an increased frequency of loop six opening and product release.

A general proof is derived that entropy production can be maximized with respect to rate constants in any enzymatic transition. This result is used to test the assumption that biological evolution of enzyme is accompanied with an increase of entropy production in its internal transitions and that such increase can serve to quantify the progress of enzyme evolution. The state of maximum entropy production would correspond to fully evolved enzyme. As an example the internal transition EP ES ↔ in a generalized reversible Michaelis-Menten three state scheme is analyzed. A good agreement is found among experimentally determined values of the forward rate constant in internal transitions EP ES → for three types of β -Lactamase enzymes and their optimal values predicted by the maximum entropy production principle, which agrees with earlier observations that β -Lactamase enzymes are nearly fully evolved. The optimization of rate constants as the consequence of basic physical principle, which is the subject of this paper, is completely different concept from a) net metabolic flux maximization or b) entropy production minimization (in the static head state), both also proposed to be tightly connected to biological evolution. .

We enzymatically deglycosylated pig lung angiotensin I-converting enzyme (ACE) to study the involvement of its glycanic chains in its physicochemical and catalytic properties. The effects of endoglycosidases F 2 and H, and of N-glycanase were assessed by ACE mobility in SDS-PAGE. N-Glycanase only was completely effective with or without previous denaturation, leading to a shift in ACE M r from 172 to 135 kDa; endoglycosidase F 2 produced the same shift but only without previous denaturation. Deglycosylated ACE had the same kcat as native ACE for the substrate hippuryl-histidyl-Ieucine, and an identical Stokes radius as measured by size-exclusion high performance liquid chromatography. Neuraminidase had no effect on ACE Stokes radius but slightly decreased its kcat which could be related to variations in ionization of the active site. The isoelectric point of ACE, as, determined by isoelectric focusing, increased from 4.5-4.8 to 5.0-53 after either endoglycosidase F 3 or neuraminidase digestion, but still with microheterogeneities which thus did not seem to be related to ACE glycans. Deglycosylated ACE did not bind onto agaroselectins in contrast to native ACE which bound strongly to concanavalin A showing interactions involving oligomannosidic or biantennary and sialylated N-acetyl-lactosaminic isoglycans. Finally, tunicamycin, an inhibitor of Nglycosylation, did not modify ACE secretion by endothelial cells. Thus, ACE glycans have no drastic effects on structural and biological properties of the protein, but they may have a functional role on intracellular targeting of both secreted and membrane-bound ACE isoforms, also for the protection of the soluble plasma form against hepatic lectins and the maintenance of its hydrosolubility.

A novel peroxidase-like artificial enzyme, named "caseoperoxidase", was biomimetically designed using a nano artificial amino acid apo-protein hydrophobic pocket. This four-component nano artificial enzyme containing heme-imidazole-β-casein-SDS exhibited high activity growth and k cat performance towards the native horseradish peroxidase (HRP) demonstrated by the steady state kinetics using UV-Vis spectrophotometry. The hydrophobicity and secondary structure of the caseoperoxidase were studied by ANS fluorescence and circular dichroism spectroscopy. Camel βcasein (Cβ-casein), with a flexible structure and exalted hydrophobicity, was selected as an appropriate apo-protein for the heme active site using a homology modeling method. Heme docking into the newly obtained Cβ-casein structure indicated one heme was mainly incorporated with Cβ-casein. The presence of a main electrostatic site for the active site in the Cβ-casein was also confirmed by experimental methods through Wyman binding potential and isothermal titration calorimetry. The existence of Cβ-casein protein in this biocatalyst lowered the suicide inactivation, and indicated that the obtained structure has a good protective role for the heme active-site. Additional further experiments confirmed the retention of caseoperoxidase structure and function as an artificial enzyme.

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

The quantitative estimation of intracellular metabolite concentrations (metabolic profiling) is a prerequisite for a better understanding of biological processes and thus inevitable for the rational improvement of microbial production strains and process design. Since pool sizes of substrates regulate flux through different enzymes, the accurate determination of intracellular metabolite concentrations is necessary to understand in vivo reaction kinetics. Quantification of intracellular concentrations of glycolytic intermediates in Escherichia coli K12 was achieved by using a novel in situ rapid sampling and quenching procedure. A new extraction procedure using buffered hot water was established. By use of simultaneous multi-substrate feeding with various ratios of glucose, fructose and acetate during continuous cultivations several metabolic states were induced. Metabolic flux analysis and the newly developed metabolic profiling procedure were used to determine in vivo enzyme kinetics as exemplified for fructose 1,6-bisphosphate aldolase and citrate synthase.

The reaction kinetics of crude firefly lantern extracts with and without added synthetic luciferin were examined. The addition of exogenous luciferin to the reaction mixture resulted in an apparent increase in net light emission per unit of ATP in solution. This additional reactivity (up to 1000-fold) enables the detection of subpicogram levels of ATP. The effects of enzyme preparation, dilution, and aging procedures on the increased sensitivity of the ATP assay, as well as the assay limitations of the crude firefly lantern extracts, are also discussed.

Polo-like kinase 1 (Plk1) is an attractive target for the development of anticancer agents due to its importance in regulating cell-cycle progression. Overexpression of Plk1 has been detected in a variety of cancers, and expression levels often correlate with poor prognosis. Despite high interest in Plk1-targeted therapeutics, there is currently no structure publicly available to guide structure-based drug design of specific inhibitors. We determined the crystal structures of the T210V mutant of the kinase domain of human Plk1 complexed with the nonhydrolyzable ATP analogue adenylylimidodiphosphate (AMPPNP) or the pyrrolo-pyrazole inhibitor PHA-680626 at 2.4 and 2.1 Å resolution, respectively. Plk1 adopts the typical kinase domain fold and crystallized in a conformation resembling the active state of other kinases. Comparison of the kinetic parameters determined for the (unphosphorylated) wild-type enzyme, as well as the T210V and T210D mutants, shows that the mutations primarily affect the k cat of the reaction, with little change in the apparent K m for the protein or nucleotide substrates (k cat ) 0.0094, 0.0376, and 0.0049 s -1 and K m(ATP) ) 3.2, 4.0, and 3.0 µM for WT, T210D, and T210V, respectively). The structure highlights features of the active site that can be exploited to obtain Plk1specific inhibitors with selectivity over other kinases and Plk isoforms. These include the presence of a phenylalanine at the bottom of the ATP pocket, combined with a cysteine (as opposed to the more commonly found leucine) in the roof of the binding site, a pocket created by Leu132 in the hinge region, and a cluster of positively charged residues in the solvent-exposed area outside of the adenine pocket adjacent to the hinge region.

We report on our efforts to create efficient artificial metalloenzymes for the enantioselective hydrogenation of N-protected dehydroamino acids using either avidin or streptavidin as host proteins. Introduction of chiral amino acid spacers -phenylalanine or proline -between the biotin anchor and the flexible aminodiphosphine moiety 1, combined with saturation mutagenesis at position S112X of streptavidin, affords second generation artificial hydrogenases displaying improved organic solvent tolerance, reaction rates (3-fold) and (S)-selectivities (up to 95 % ee for N-acetamidoalanine and N-acetamidophenylalanine). It is shown that these artificial metalloenzymes follow Michaelis-Menten kinetics with an increased affinity for the substrate and a higher k cat than the protein-free catalyst (compare k cat 3.06 min À1 and K M 7.38 mM for [RhA C H T U N G T R E N N U N G (COD)Biot-1] + with k cat 12.30 min À1 and K M 4.

At high cellobiose concentrations, the cellobiase activity of a Trichoderma reesei cellulase preparation does not follow Michaelis-Menten kinetics and shows substrate inhibition. Several rate equations were fitted to the initial rate-cellobiose concentration data. The best fit is obtained for a rate equation corresponding to partial substrate inhibition of cellobiase. In this case, the K m , V max and K I values obtained are 1.1 mM, 16 IU ml -1 and 26 mM, respectively.

Objectives: While nitric oxide (NO) is a potent vasodilator already in the nM range, a cGMP-independent negative inotropic effect is observed at higher concentrations. Since inhibition of creatine kinase (CK) by NO-induced nitrosylation has been proposed as a possible 31 mechanism of action, we measured the flux through CK in the intact heart. Methods: In saline perfused, paced guinea pig hearts P NMR spectroscopy was employed to directly assess the cardiac energy status, i.e. free energy of ATP hydrolysis (DG ) and flux ATP through CK using magnetization transfer in absence and presence of NO. Results: NO (50 mM) doubled coronary flow and induced a rapid drop in left ventricular developed pressure (39610 vs. 81610 mmHg) and MVO (1.360.8 vs. 3.760.5 mmol / min / g) (n57). This 2 effect was associated with an immediate decrease in phosphocreatine (PCr) (269%) and DG . During the subsequent 35 min of NO ATP infusion cardiac function and MVO remained depressed, while PCr partially recovered. NO had no effect on the unidirectional forward 2 flux through CK (98621 vs. 99620 mmol / min / g, n57) which was 5-to 10-fold greater than the rate of ATP turnover. Upon cessation of NO infusion both cardiac function and PCr rapidly returned to baseline values. The NO-induced fall in the myocardial energy status was associated with an increase in mitochondrial NADH (n57) as assessed by surface fluorescence. The observed change in fluorescence was similar to that observed with short term ischemia. Conclusion: The NO-mediated depression of myocardial function, MVO and 2 energy status is not mediated by changes in CK flux. Most likely a partial blockade of mitochondrial oxidative phosphorylation at the level of cytochrome c oxidase is responsible for this effect.

Nucleotide analogues that incorporate a metabolically labile nucleoside phosphoramidate (a ProTide) have found utility as prodrugs. In humans, ProTides can be cleaved by human histidine triad nucleotide binding protein 1 (hHint1) to expose the nucleotide monophosphate. Activation by this route circumvents highly selective nucleoside kinases that limit the use of nucleosides as prodrugs. To better understand the diversity of potential substrates of hHint1, we created and studied a series of phosphoramidate nucleosides. Using a combination of enzyme kinetics, X-ray crystallography, and isothermal titration calorimetry with both wild-type and inactive mutant enzymes, we have been able to explore the energetics of substrate binding and establish a structural basis for catalytic efficiency. Diverse nucleobases are well tolerated, but portions of the ribose are needed to position substrates for catalysis. Beneficial characteristics of the amine leaving group are also revealed. Structural ...