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Malcolm D Walkinshaw - One of the best experts on this subject based on the ideXlab platform.
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the kinetic characteristics of human and trypanosomatid phosphofructokinases for the Reverse Reaction
Biochemical Journal, 2019Co-Authors: Peter M Fernandes, James Kinkead, Frédéric Bringaud, Iain W. Mcnae, Paul A M Michels, Malcolm D WalkinshawAbstract:Eukaryotic ATP-dependent phosphofructokinases (PFKs) are often considered unidirectional enzymes catalysing the transfer of a phospho moiety from ATP to fructose 6-phosphate (F6P) to produce ADP and fructose 1,6-bisphosphate (F16BP). The Reverse Reaction is not generally considered to occur under normal conditions and has never been demonstrated for any eukaryotic ATP-dependent PFKs, though it does occur in PPi-dependent PFKs and has been experimentally shown for bacterial ATP-dependent PFKs. Evidence is provided via two orthogonal assays that all three human PFK isoforms can catalyse the Reverse Reaction in vitro , allowing determination of kinetic properties. Additionally, the Reverse Reaction was shown possible for PFKs from three clinically important trypanosomatids; these enzymes are contained within glycosomes in vivo . This compartmentalisation may facilitate reversal, given the potential for trypanosomatids to have an altered ATP/ADP ratio in glycosomes compared to the cytosol. The kinetic properties of each trypanosomatid PFK were determined, including the response to natural and artificial modulators of enzyme activity. The possible physiological relevance of the Reverse Reaction in trypanosomatid and human PFKs is discussed.
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The kinetic characteristics of human and trypanosomatid phosphofructokinases for the Reverse Reaction.
Biochemical Journal, 2019Co-Authors: Peter M Fernandes, James Kinkead, Frédéric Bringaud, Iain W. Mcnae, Paul A M Michels, Malcolm D WalkinshawAbstract:Eukaryotic ATP-dependent phosphofructokinases (PFKs) are often considered unidirectional enzymes catalysing the transfer of a phospho moiety from ATP to fructose 6-phosphate to produce ADP and fructose 1,6-bisphosphate. The Reverse Reaction is not generally considered to occur under normal conditions and has never been demonstrated for any eukaryotic ATP-dependent PFKs, though it does occur in inorganic pyrophosphate-dependent PFKs and has been experimentally shown for bacterial ATP-dependent PFKs. The evidence is provided via two orthogonal assays that all three human PFK isoforms can catalyse the Reverse Reaction in vitro, allowing determination of kinetic properties. Additionally, the Reverse Reaction was shown possible for PFKs from three clinically important trypanosomatids; these enzymes are contained within glycosomes in vivo. This compartmentalisation may facilitate reversal, given the potential for trypanosomatids to have an altered ATP/ADP ratio in glycosomes compared with the cytosol. The kinetic properties of each trypanosomatid PFK were determined, including the response to natural and artificial modulators of enzyme activity. The possible physiological relevance of the Reverse Reaction in trypanosomatid and human PFKs is discussed.
Peter M Fernandes - One of the best experts on this subject based on the ideXlab platform.
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the kinetic characteristics of human and trypanosomatid phosphofructokinases for the Reverse Reaction
Biochemical Journal, 2019Co-Authors: Peter M Fernandes, James Kinkead, Frédéric Bringaud, Iain W. Mcnae, Paul A M Michels, Malcolm D WalkinshawAbstract:Eukaryotic ATP-dependent phosphofructokinases (PFKs) are often considered unidirectional enzymes catalysing the transfer of a phospho moiety from ATP to fructose 6-phosphate (F6P) to produce ADP and fructose 1,6-bisphosphate (F16BP). The Reverse Reaction is not generally considered to occur under normal conditions and has never been demonstrated for any eukaryotic ATP-dependent PFKs, though it does occur in PPi-dependent PFKs and has been experimentally shown for bacterial ATP-dependent PFKs. Evidence is provided via two orthogonal assays that all three human PFK isoforms can catalyse the Reverse Reaction in vitro , allowing determination of kinetic properties. Additionally, the Reverse Reaction was shown possible for PFKs from three clinically important trypanosomatids; these enzymes are contained within glycosomes in vivo . This compartmentalisation may facilitate reversal, given the potential for trypanosomatids to have an altered ATP/ADP ratio in glycosomes compared to the cytosol. The kinetic properties of each trypanosomatid PFK were determined, including the response to natural and artificial modulators of enzyme activity. The possible physiological relevance of the Reverse Reaction in trypanosomatid and human PFKs is discussed.
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The kinetic characteristics of human and trypanosomatid phosphofructokinases for the Reverse Reaction.
Biochemical Journal, 2019Co-Authors: Peter M Fernandes, James Kinkead, Frédéric Bringaud, Iain W. Mcnae, Paul A M Michels, Malcolm D WalkinshawAbstract:Eukaryotic ATP-dependent phosphofructokinases (PFKs) are often considered unidirectional enzymes catalysing the transfer of a phospho moiety from ATP to fructose 6-phosphate to produce ADP and fructose 1,6-bisphosphate. The Reverse Reaction is not generally considered to occur under normal conditions and has never been demonstrated for any eukaryotic ATP-dependent PFKs, though it does occur in inorganic pyrophosphate-dependent PFKs and has been experimentally shown for bacterial ATP-dependent PFKs. The evidence is provided via two orthogonal assays that all three human PFK isoforms can catalyse the Reverse Reaction in vitro, allowing determination of kinetic properties. Additionally, the Reverse Reaction was shown possible for PFKs from three clinically important trypanosomatids; these enzymes are contained within glycosomes in vivo. This compartmentalisation may facilitate reversal, given the potential for trypanosomatids to have an altered ATP/ADP ratio in glycosomes compared with the cytosol. The kinetic properties of each trypanosomatid PFK were determined, including the response to natural and artificial modulators of enzyme activity. The possible physiological relevance of the Reverse Reaction in trypanosomatid and human PFKs is discussed.
M Ozeir - One of the best experts on this subject based on the ideXlab platform.
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structural basis for substrate selectivity and nucleophilic substitution mechanisms in human adenine phosphoribosyltransferase catalyzed Reaction
Journal of Biological Chemistry, 2019Co-Authors: M Ozeir, J Huyet, Marieclaude Burgevin, Francoise Chesney, Jeanmarc Remy, Abdul Rauf Siddiqi, Benoit Pinson, Roland LupoliAbstract:The reversible adenine phosphoribosyltransferase enzyme (APRT) is essential for purine homeostasis in prokaryotes and eukaryotes. In humans, APRT (hAPRT) is the only enzyme known to produce AMP in cells from dietary adenine. APRT can also process adenine analogs, which are involved in plant development or neuronal homeostasis. However, the molecular mechanism underlying substrate specificity of APRT and catalysis in both directions of the Reaction remains poorly understood. Here we present the crystal structures of hAPRT complexed to three cellular nucleotide analogs (hypoxanthine, IMP, and GMP) that we compare with the phosphate-bound enzyme. We established that binding to hAPRT is substrate shape-specific in the forward Reaction, whereas it is base-specific in the Reverse Reaction. Furthermore , a quantum mechanics/molecular mechanics (QM/ MM) analysis suggests that the forward Reaction is mainly a nucleophilic substitution of type 2 (S N 2) with a mix of S N 1-type molecular mechanism. Based on our structural analysis, a magnesium-assisted S N 2-type mechanism would be involved in the Reverse Reaction. These results provide a framework for understanding the molecular mechanism and substrate discrimination in both directions by APRTs. This knowledge can play an instrumental role in the design of inhibitors, such as antiparasitic agents, or adenine-based substrates.
Kohji Mitsubayashi - One of the best experts on this subject based on the ideXlab platform.
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fiber optic bio sniffer biochemical gas sensor using Reverse Reaction of alcohol dehydrogenase for exhaled acetaldehyde
ACS Sensors, 2018Co-Authors: Kenta Iitani, Pojen Chien, Takuma Suzuki, Koji Toma, Takahiro Arakawa, Yasuhiko Iwasaki, Kohji MitsubayashiAbstract:Volatile organic compounds (VOCs) exhaled in breath have huge potential as indicators of diseases and metabolisms. Application of breath analysis for disease screening and metabolism assessment is expected since breath samples can be noninvasively collected and measured. In this research, a highly sensitive and selective biochemical gas sensor (bio-sniffer) for gaseous acetaldehyde (AcH) was developed. In the AcH bio-sniffer, a Reverse Reaction of alcohol dehydrogenase (ADH) was employed for reducing AcH to ethanol and simultaneously consuming a coenzyme, reduced form of nicotinamide adenine dinucleotide (NADH). The concentration of AcH can be quantified by fluorescence detection of NADH that was consumed by Reverse Reaction of ADH. The AcH bio-sniffer was composed of an ultraviolet light-emitting diode (UV-LED) as an excitation light source, a photomultiplier tube (PMT) as a fluorescence detector, and an optical fiber probe, and these three components were connected with a bifurcated optical fiber. A gas...
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Fiber-Optic Bio-sniffer (Biochemical Gas Sensor) Using Reverse Reaction of Alcohol Dehydrogenase for Exhaled Acetaldehyde.
ACS Sensors, 2018Co-Authors: Kenta Iitani, Pojen Chien, Takuma Suzuki, Koji Toma, Takahiro Arakawa, Yasuhiko Iwasaki, Kohji MitsubayashiAbstract:Volatile organic compounds (VOCs) exhaled in breath have huge potential as indicators of diseases and metabolisms. Application of breath analysis for disease screening and metabolism assessment is expected since breath samples can be noninvasively collected and measured. In this research, a highly sensitive and selective biochemical gas sensor (bio-sniffer) for gaseous acetaldehyde (AcH) was developed. In the AcH bio-sniffer, a Reverse Reaction of alcohol dehydrogenase (ADH) was employed for reducing AcH to ethanol and simultaneously consuming a coenzyme, reduced form of nicotinamide adenine dinucleotide (NADH). The concentration of AcH can be quantified by fluorescence detection of NADH that was consumed by Reverse Reaction of ADH. The AcH bio-sniffer was composed of an ultraviolet light-emitting diode (UV-LED) as an excitation light source, a photomultiplier tube (PMT) as a fluorescence detector, and an optical fiber probe, and these three components were connected with a bifurcated optical fiber. A gas...
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Improved Sensitivity of Acetaldehyde Biosensor by Detecting ADH Reverse Reaction-Mediated NADH Fluoro-Quenching for Wine Evaluation
ACS Sensors, 2017Co-Authors: Kenta Iitani, Pojen Chien, Takuma Suzuki, Koji Toma, Takahiro Arakawa, Yasuhiko Iwasaki, Kohji MitsubayashiAbstract:Acetaldehyde (AcH) is found in ambient air, foods, and the living body. This toxic substance is also contained in wine and known as an important ingredient affecting the quality of wine. Herein, we constructed and evaluated two different fiber-optic biosensors for measurement of AcH in the liquid phase (AcH biosensor) using aldehyde dehydrogenase (ALDH) or alcohol dehydrogenase (ADH). The AcH biosensor measured a concentration of AcH using fluorescence intensity of a reduced form of nicotinamide adenine dinucleotide (NADH) that was produced or consumed via catalytic Reaction of the respective enzyme. In the AcH measurement system, an ultraviolet light emitting diode (UV-LED) and photomultiplier tube (PMT) were connected to a bifurcated optical fiber and were used to excite and detect NADH. A sensing region was developed using an optical fiber probe and an enzyme-immobilized membrane, buffer pH, and concentrations of a coenzyme in buffer solution for ALDH forward Reaction and ADH Reverse Reaction were opti...
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Fluorometric gas-imaging system (sniff-cam), using the extinction of NADH with an ADH Reverse Reaction, for acetaldehyde in the gas phase
The Analyst, 2017Co-Authors: Kenta Iitani, Koji Toma, Takahiro Arakawa, Toshiyuki Sato, Munire Naisierding, Yuuki Hayakawa, Kohji MitsubayashiAbstract:A gas-imaging system (sniff-cam) that allows fluorometric visualization of a two-dimensional (2-D) distribution of gaseous acetaldehyde (AcH) was developed. It employed a Reverse Reaction of a nicotinamide adenine dinucleotide (NADH) dependent enzyme that led to consumption of NADH in that Reaction. The system was constructed with a highly sensitive camera, an ultraviolet light emitting diode array sheet, two band pass filters and an alcohol dehydrogenase (ADH)-immobilized mesh that was used for AcH detection. The Reverse Reaction of the ADH catalyzed the reduction of AcH to ethanol and the oxidation of NADH to NAD+, which occurred when gaseous AcH was applied to the ADH immobilized mesh that was wetted with a slightly acidic NADH solution. As NADH has an autofluorescence property [emission (λem) at 490 nm; excitation (λex) at 340 nm], the presence of gaseous AcH was visualized by a decrease of fluorescence of the NADH at the ADH immobilized mesh. After constructing the gaseous AcH imaging system, optimizations of pH, and concentration of the NADH solution were performed. As a result of the optimizations (500 μM of NADH in 0.1 M of Tris hydrochloride (Tris-HCl) buffer at pH 6.5), the AcH sniff-cam showed a wide dynamic range (0.1–10 ppm) for gaseous AcH with a high correlation coefficient (R = 0.999). Furthermore, a fluorescence gradient with a rounded shape centered in a gas outlet was observed. These results demonstrated that the AcH sniff-cam utilizing the fluorescence decrease of NADH could be used to quantitatively evaluate the 2-D distribution of gaseous AcH.
Byung-gee Kim - One of the best experts on this subject based on the ideXlab platform.
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Kinetic modeling of ??-transamination for enzymatic kinetic resolution of ??-methylbenzylamine
Biotechnology and Bioengineering, 1998Co-Authors: Jong Shik Shin, Byung-gee KimAbstract:A kinetic model for ω-transaminase from Bacillus thuringiensis JS64 was developed by using the King-Altman method to simulate the kinetic resolution of α-methylbenzylamine (α-MBA). Starting from a ping-pong bi-bi mechanism, a complete kinetic model including substrate inhibition only in the Reverse Reaction (i.e., transamination between acetophenone and L-alanine) was developed. The asymmetric synthesis of (S)-α-MBA proved to be difficult due to a much lower maximum Reverse Reaction rate than the maximum forward Reaction rate, thermodynamically exergonic forward Reaction (i.e., transamination between (S)-α-MBA and pyruvate), and the severe product and substrate inhibition of the Reverse Reaction. Experimental values for kinetic parameters show that the product inhibition constant of (S)-α-MBA is the most important parameter on determining the resolution Reaction rate, suggesting that the resolution Reaction rate will be very low unless (S)-α-MBA strongly inhibits the Reverse Reaction. Using the kinetic model, the kinetic resolution of α-MBA in aqueous buffer was simulated, and the simulation results showed a high degree of consistency with experimental data over a range of Reaction conditions. Various simulation results suggest that the crucial bottleneck in the kinetic resolution of α-MBA lies mainly in the accumulation of acetophenone in Reaction media as the Reaction proceeds, whereas L-alanine exerts a little inhibitory effect on the Reaction. The model predicts that removing acetophenone produced during the Reaction can enhance the Reaction rate dramatically. Indeed, the biphasic Reaction system is capable of extracting acetophenone from the aqueous phase, showing a much higher Reaction rate compared to a monophasic Reaction system. The kinetic model was also useful in predicting the properties of other, better enzymes as well as the optimal concentrations of amino acceptor and enzyme in the resolution Reaction. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 534–540, 1998.
