Michaelis-Menten Rate Constants Research Articles

A genomic database furnishes minimal functional glycyl-tRNA synthetases homologous to other, designed class II urzymes.

The hypothesis that conserved core catalytic sites could represent ancestral aminoacyl-tRNA synthetases (AARS) drove the design of functional TrpRS, LeuRS, and HisRS 'urzymes'. We describe here new urzymes detected in the genomic record of the arctic fox, Vulpes lagopus. They are homologous to the α-subunit of bacterial heterotetrameric Class II glycyl-tRNA synthetase (GlyRS-B) enzymes. AlphaFold2 predicted that the N-terminal 81 amino acids would adopt a 3D structure nearly identical to our designed HisRS urzyme (HisCA1). We expressed and purified that N-terminal segment and the spliced open reading frame GlyCA1-2. Both exhibit robust single-turnover burst sizes and ATP consumption rates higher than those previously published for HisCA urzymes and comparable to those for LeuAC and TrpAC. GlyCA is more than twice as active in glycine activation by adenosine triphosphate as the full-length GlyRS-B α2 dimer. Michaelis-Menten rate constants for all three substrates reveal significant coupling between Exon2 and both substrates. GlyCA activation favors Class II amino acids that complement those favored by HisCA and LeuAC. Structural features help explain these results. These minimalist GlyRS catalysts are thus homologous to previously described urzymes. Their properties reinforce the notion that urzymes may have the requisite catalytic activities to implement a reduced, ancestral genetic coding alphabet.

Counter Anion-Directed Growth of Iron Oxide Nanorods in a Polyol Medium with Efficient Peroxidase-Mimicking Activity for Degradation of Dyes in Contaminated Water.

Development of nanozymes, which are nanomaterials with intrinsic enzymatic properties, has emerged as an appealing alternative to the natural enzymes with tremendous application potential from the chemical industry to biomedicine. The self-assembled growth of micrometer-sized oxide materials with controlled nonspherical shapes can be an important tool for enhancing activity as artificial enzymes, as the formation of these superstructures often results in high surface area with favorable impact on catalytic activity. Herein, the growth of rod-shaped Fe3O4 microstructures via a one-pot microwave-based method and using a water–poly(ethylene glycol) mixture as a solvent is reported, without the involvement of external shape-directing agents. The precursor metal salt played a key role in the size, shape, and phase selective evolution of iron oxide micro/nanomaterials. Whereas self-assembled microrod superstructures were obtained using Fe(NO3)3 as the metal salt precursor, use of FeCl3 or Fe-acetate as precursors afforded hollow Fe2O3 microparticles and Fe3O4 nanoparticles, respectively. A graphitic layer was deposited on the Fe3O4 surface, imparting a negative surface charge as a result of a high-temperature treatment of poly(ethylene glycol). The rod-shaped Fe3O4 microcrystals show efficient peroxidase-mimicking activity toward 3,3,5,5′-tetramethylbenzidine and pyrogallol as peroxidase substrates with a Michaelis–Menten rate constant (Km) value of 0.05 and 0.52 mM, respectively. The proficient enzyme mimicking behavior of these magnetic superstructures was further explored for the degradation of organic dyes that includes rhodamine B, methylene blue, and methyl orange with a rate constant (k) of 0.038, 0.011, and 0.007 min–1 respectively, using H2O2. This fast and simple method could help to develop a new pathway for differently shaped oxide nanoparticles in a sustainable and economical manner that can be harnessed as nanozymes for industrial as well as biological applications.

A Nanostructured Bifunctional platform for Sensing of Glucose Biomarker in Artificial Saliva: Synergy in hybrid Pt/Au surfaces

We report on a bimetallic, bifunctional electrode where a platinum (Pt) surface was patterned with nanostructured gold (Au) fingers with different film thicknesses, which was functionalized with glucose oxidase (GOx) to yield a highly sensitive glucose biosensor. This was achieved by using selective adsorption of a self-assembled monolayer (SAM) onto Au fingers, which allowed GOx immobilization only onto the Au-SAM surface. This modified electrode was termed bifunctional because it allowed to simultaneously immobilize the biomolecule (GOx) on gold to catalyze glucose, and detect hydrogen peroxide on Pt sites. Optimized electrocatalytic activity was reached for the architecture Pt/Au-SAM/GOx with 50nm thickness of Au, where synergy between Pt and Au allowed for detection of hydrogen peroxide (H2O2) at a low applied potential (0V vs. Ag/AgCl). Detection was performed for H2O2 in the range between 4.7 and 102.7 nmol L–1, with detection limit of 3.4×10−9 mol L−1 (3.4 nmol L−1) and an apparent Michaelis-Menten rate constant of 3.2×10−6molL−1, which is considerably smaller than similar devices with monometallic electrodes. The methodology was validated by measuring glucose in artificial saliva, including in the presence of interferents. The synergy between Pt and Au was confirmed in electrochemical impedance spectroscopy measurements with an increased electron transfer, compared to bare Pt and Au electrodes. The approach for fabricating the reproducible bimetallic Pt/Au electrodes is entirely generic and may be explored for other types of biosensors and biodevices where advantage can be taken of the combination of the two metals.

Application of oxo-manganese complex immobilized on ion-exchange polymeric film as biomimetic sensor for nitrite ions

A biomimetic sensor based on oxo-bridged dinuclear manganese–phenanthroline complex immobilized into an ion-exchange polymeric film deposited on glassy carbon electrode was applied to detection of nitrite ions and studied according to their kinetics parameters. The cyclic voltammetry at the modified electrode in universal buffer showed a two electron oxidation/reduction of the couple MnIV(μ-O)2MnIV/MnIII(μ-O)2MnIII and electrocatalytic property toward nitrite oxidation with a decrease of the overpotential of 320mV compared with the bare glassy carbon electrode. A plot of the anodic current vs. the nitrite ions concentration for potential fixed (+0.480) at the biomimetic sensor was linear in the 2.49×10−6 to 9.90×10−6molL−1 concentration range with a detection limit of 6.50×10−6molL−1. The kinetic mechanism was derived by Michaelis–Menten, then, kinetics parameters were calculated through four methods: Lineweaver–Burke, Eadie–Hofstee, Hanes–Woolf and Nonlinear curve fitting. The best results were Michaelis–Menten rate constant=3.42μmolL−1, catalytic rate constant=0.0114s−1, catalytic efficiency=3.3×103(molL)−1s−1 and heterogeneous electrochemical rate constant=1.15×10−5cms−1.

In vitro Metabolic Investigational Studies of Herbal-Drug Interactions Leading to Predictive Clinical Outcomes

One main area of our research focuses on the impact of herbal supplements or their individual components on the enzymatic bioactivation of chemotherapeutic drugs. Previous studies have found that over half of the patients that report using herbal supplements are cancer patients and that over 72% of them do not inform their healthcare provider about their herbal supplement use [2-5]. The risk of potential unwanted interactions is increased for chemotherapeutic drugs because of the narrow therapeutic index that most of these drugs have. Therefore, a relatively small change in systemic exposure of the drug as a result of an unwanted drug–herbal supplement interaction could result in either sub-therapeutic effects or undesired serious side effects. In this area we have focused much of our effort on two chemotherapeutic prodrugs which are converted to active metabolites by different enzymatic systems. The first, tamoxifen, relies heavily on various cytochrome P450 enzymes to form its active metabolites 4-hydroxytamoxifen and endoxifen, while the second drug, irinotecan, utilizes carboxyesterases to form its chemoactive metabolite SN-38. Our laboratory utilizes in-vitro metabolic inhibition and LCtandem mass spectrometry to determine the impact on the metabolic reaction velocities as a function of herbal supplement concentration. The experiments are designed to obtain data such as IC 50 values of herbal supplements on the various metabolic pathways of the prodrugs, inhibition kinetics and mode of inhibition. After a thorough characterization of each drug in the absence of any inhibitor with our microsomal system, we selected optimal reaction conditions for each substrate evaluated. Substrate concentrations are based on physiologically relevant doses as well as concentrations that are near or below the previously measured Michaelis-Menten rate constant (K m ) values. We then conducted a broad range finding study where the concentration of the herbal supplements is varied over a large range to determine what degree of inhibition is occurring. If inhibition of the reaction velocity is found to be less than 50% at the upper range of herbal concentrations tested, the herbal supplement is considered not to be a significant inhibitor and is not studied further as these upper concentration ranges exceed a typical physiological exposure. Each herbal that produced more than 50% inhibition in reaction velocity over this range is further studied at lower herbal concentrations and various substrate concentrations to determine their IC 50 and other kinetic values. Additionally we also conduct a compositional analysis on these herbal supplements found to produce inhibition using HPLCDAD and LC-MS/MS in order to further investigate the effects of the individual components of the herbals as well. Our laboratory has recently completed broad range screening experiments for ten commonly used herbal supplements (Echinacea, Ginseng, Skullcap, Lemon balm, Licorice Root, Feverfew, Burdock Root, Turmeric, Valerian Root, Gingko Leaf) that are concomitantly taken with either tamoxifen or irinotecan chemotherapy. The screening studies revealed that two of these herbals (Skullcap, Lemon balm) strongly inhibited the cytochrome P450 metabolic activation pathway of tamoxifen while two others (Echinacea, Ginseng) were determined to be less potent inhibitors based on their IC 50 values. For irinotecan only, Skullcap and Lemon balm were found to inhibit the carboxyesterase bioactivation pathway. Evaluation of the measured IC 50 values expressed as a percent of a single oral dose for tamoxifen ranged from 0.02 to 0.6% while the values for irinotecan ranged from 0.21 to 0.25%. This data suggests that these herbals could potentially results in sub-therapeutic levels of active metabolites. Furthermore, LineweaverBurke plots of the kinetic data suggest that the mode of inhibition for both herbals with irinotecan was competitive while a non-competitive mode was evident for 4-hydroxytamoxifen and an indeterminate mode was observed for the endoxifen metabolite. Previously, our laboratory has shown the impact of St. John’s Wort, Ginger and Black Cohosh on these same chemotherapeutic prodrugs [6]. This data suggests that black cohosh was the most potent inhibitor of the bioactivation of both tamoxifen and irinotecan, while ginger and St. John’s Wort were observed to be more potent for CYP450 mediated pathways of tamoxifen than carboxyesterase activation pathways of irinotecan. Other commonly used herbal supplements evaluated include milk thistle with tamoxifen, which showed relatively weak inhibition of 4-hydroxytamoxifen via a noncompetitive mode and strong inhibition of the endoxifen metabolite which had an indeterminate mode of inhibition due to significant levels of inhibition

Enzymatic Reactions in Microfluidic Devices: Michaelis−Menten Kinetics

Kinetic rate constants for enzymatic reactions are typically measured with a series of experiments at different substrate concentrations in a well-mixed container. Here we demonstrate a microfluidic technique for measuring Michaelis-Menten rate constants with only a single experiment. Enzyme and substrate are brought together in a coflow microfluidic device, and we establish analytically and numerically that the initial concentration of product scales with the distance x along the channel as x5/2. Measurements of the initial rate of product formation, combined with the quasi-steady rate of product formation further downstream, yield the rate constants. We corroborate the x5/2 scaling result experimentally using the bioluminescent reaction between ATP and luciferase/luciferin as a model system.

Characterization of deltamethrin metabolism by rat plasma and liver microsomes

Deltamethrin, a widely used type II pyrethroid insecticide, is a relatively potent neurotoxicant. While the toxicity has been extensively examined, toxicokinetic studies of deltamethrin and most other pyrethroids are very limited. The aims of this study were to identify, characterize, and assess the relative contributions of esterases and cytochrome P450s (CYP450s) responsible for deltamethrin metabolism by measuring deltamethrin disappearance following incubation of various concentrations (2 to 400 microM) in plasma (esterases) and liver microsomes (esterases and CYP450s) prepared from adult male rats. While the carboxylesterase metabolism in plasma and liver was characterized using an inhibitor, tetra isopropyl pyrophosphoramide (isoOMPA), CYP450 metabolism was characterized using the cofactor, NADPH. Michaelis-Menten rate constants were calculated using linear and nonlinear regression as applicable. The metabolic efficiency of these pathways was estimated by calculating intrinsic clearance (Vmax/Km). In plasma, isoOMPA completely inhibited deltamethrin biotransformation at concentrations (2 and 20 microM of deltamethrin) that are 2- to 10-fold higher than previously reported peak blood levels in deltamethrin-poisoned rats. For carboxylesterase-mediated deltamethrin metabolism in plasma, Vmax=325.3+/-53.4 nmol/h/ml and Km=165.4+/-41.9 microM. Calcium chelation by EGTA did not inhibit deltamethrin metabolism in plasma or liver microsomes, indicating that A-esterases do not metabolize deltamethrin. In liver microsomes, esterase-mediated deltamethrin metabolism was completely inhibited by isoOMPA, confirming the role of carboxylesterases. The rate constants for liver carboxylesterases were Vmax=1981.8+/-132.3 nmol/h/g liver and Km=172.5+/-22.5 microM. Liver microsomal CYP450-mediated biotransformation of deltamethrin was a higher capacity (Vmax=2611.3+/-134.1 nmol/h/g liver) and higher affinity (Km=74.9+/-5.9 microM) process than carboxylesterase (plasma or liver) detoxification. Genetically engineered individual rat CYP450s (Supersomes) were used to identify specific CYP450 isozyme(s) involved in the deltamethrin metabolism. CYP1A2, CYP1A1, and CYP2C11 in decreasing order of importance quantitatively, metabolized deltamethrin. Intrinsic clearance by liver CYP450s (35.5) was more efficient than that by liver (12.0) or plasma carboxylesterases (2.4).

The Metabolic Rate Constants and Specific Activity of Human and Rat Hepatic Cytochrome P-450 2E1 Toward Toluene and Chloroform

Chloroform (CHCl3) is a near-ubiquitous environmental contaminant, a by-product of the disinfection of drinking water sources and a commercially important compound. Standards for safe exposure have been established based on information defining its toxicity, which is mediated by metabolites. The metabolism of CHCl3 is via cytochrome P-450 2E1 (CYP2E1)-mediated oxidation to phosgene, which is known to obey a saturable mechanism. CYP2E1 is a highly conserved form, expressed in all mammalian systems studied, and is responsible for the metabolism of a great many low-molecular-weight (halogenated) compounds. However, the Michaelis–Menten rate constants for CHCl3 oxidation have not been derived in vitro, and the specific activity of CYP2E1 toward CHCl3 has not been reported. In this investigation with microsomal protein (MSP), apparent Vmax values of 27.6 and 28.3 nmol/h/mg MSP and apparent Km values of 1 and 0.15 μM in rats and human organ donors, respectively, were demonstrated. The specific activity of CYP2E1 toward CHCl3 in rats and humans was 5.29 and 5.24 pmol/min/pmol CYP2E1, respectively. Toluene metabolism to benzyl alcohol (BA), another CYP2E1-dependent reaction, was also highly dependent on CYP2E1 content in humans, and was more efficient than was CHCl3 metabolism. The specific activity of human CYP2E1 toward toluene metabolism in human MSP was 23 pmol/min/pmol CYP2E1. These results demonstrate that differences in CYP2E1 content of MSP among individuals and between species are largely responsible for observed differences in toluene and CHCl3 metabolism in vitro.

Basic biochemical laboratory procedures and computers

A biomimetic sensor based on oxo-bridged dinuclear manganese–phenanthroline complex immobilized into an ion-exchange polymeric film deposited on glassy carbon electrode was applied to detection of nitrite ions and studied according to their kinetics parameters. The cyclic voltammetry at the modified electrode in universal buffer showed a two electron oxidation/reduction of the couple MnIV(μ-O)2MnIV/MnIII(μ-O)2MnIII and electrocatalytic property toward nitrite oxidation with a decrease of the overpotential of 320 mV compared with the bare glassy carbon electrode. A plot of the anodic current vs. the nitrite ions concentration for potential fixed (+0.480) at the biomimetic sensor was linear in the 2.49 × 10−6 to 9.90 × 10−6 mol L−1 concentration range with a detection limit of 6.50 × 10−6 mol L−1. The kinetic mechanism was derived by Michaelis–Menten, then, kinetics parameters were calculated through four methods: Lineweaver–Burke, Eadie–Hofstee, Hanes–Woolf and Nonlinear curve fitting. The best results were Michaelis–Menten rate constant = 3.42 μmol L−1, catalytic rate constant = 0.0114 s−1, catalytic efficiency = 3.3 × 103 (mol L)−1 s−1 and heterogeneous electrochemical rate constant = 1.15 × 10−5 cm s−1.