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1
Guerrieri, Antonio, Rosanna Ciriello, Giuliana Bianco, Francesca De Gennaro, and Silvio Frascaro. "Allosteric Enzyme-Based Biosensors—Kinetic Behaviours of Immobilised L-Lysine-α-Oxidase from Trichoderma viride: pH Influence and Allosteric Properties." Biosensors 10, no. 10 (October 17, 2020): 145. http://dx.doi.org/10.3390/bios10100145.
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The present study describes the kinetics of L-lysine-α-oxidase (LO) from Trichoderma viride immobilised by co-crosslinking onto the surface of a Pt electrode. The resulting amperometric biosensor was able to analyse L-lysine, thus permitting a simple but thorough study of the kinetics of the immobilised enzyme. The kinetic study evidenced that LO behaves in an allosteric fashion and that cooperativity is strongly pH-dependent. Not less important, experimental evidence shows that cooperativity is also dependent on substrate concentration at high pH and behaves as predicted by the Monod-Wyman-Changeux model for allosteric enzymes. According to this model, the existence of two different conformational states of the enzyme was postulated, which differ in Lys species landing on LO to form the enzyme–substrate complex. Considerations about the influence of the peculiar LO kinetics on biosensor operations and extracorporeal reactor devices will be discussed as well. Not less important, the present study also shows the effectiveness of using immobilised enzymes and amperometric biosensors not only for substrate analysis, but also as a convenient tool for enzyme kinetic studies.
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Hochendoner, Philip, Curtis Ogle, and William H. Mather. "A queueing approach to multi-site enzyme kinetics." Interface Focus 4, no. 3 (June 6, 2014): 20130077. http://dx.doi.org/10.1098/rsfs.2013.0077.
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Multi-site enzymes, defined as where multiple substrate molecules can bind simultaneously to the same enzyme molecule, play a key role in a number of biological networks, with the Escherichia coli protease ClpXP a well-studied example. These enzymes can form a low latency ‘waiting line’ of substrate to the enzyme's catalytic core, such that the enzyme molecule can continue to collect substrate even when the catalytic core is occupied. To understand multi-site enzyme kinetics, we study a discrete stochastic model that includes a single catalytic core fed by a fixed number of substrate binding sites. A natural queueing systems analogy is found to provide substantial insight into the dynamics of the model. From this, we derive exact results for the probability distribution of the enzyme configuration and for the distribution of substrate departure times in the case of identical but distinguishable classes of substrate molecules. Comments are also provided for the case when different classes of substrate molecules are not processed identically.
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Pyne, N. J., M. E. Cooper, and M. D. Houslay. "Identification and characterization of both the cytosolic and particulate forms of cyclic GMP-stimulated cyclic AMP phosphodiesterase from rat liver." Biochemical Journal 234, no. 2 (March 1, 1986): 325–34. http://dx.doi.org/10.1042/bj2340325.
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Two enzymes displaying cyclic GMP-stimulated cyclic AMP phosphodiesterase activity were purified from rat liver to apparent homogeneity: a ‘particulate enzyme’ found as an integral membrane protein associated with the plasma membrane, and a ‘soluble’ enzyme found in the cytosol. The physical properties of these enzymes were very similar, being dimers of Mr 134,000, composed in each instance of two subunits of Mr = 66,000-67,000. Both enzymes showed similar kinetics for cyclic AMP hydrolysis. They are both high-affinity enzymes, with kinetic constants for the particulate enzyme of Km = 34 microM and Vmax. = 4.0 units/mg of protein and for the cytosolic enzyme Km = 40 microM and Vmax. = 4.8 units/mg of protein. In both instances hydrolysis of cyclic AMP appeared to show apparent positive co-operativity, with Hill coefficients (happ.) of 1.5 and 1.6 for the particulate and cytosolic enzymes respectively. However, in the presence of 2 microM-cyclic GMP, the hydrolysis of cyclic AMP obeyed Michaelis kinetics (happ. = 1) for both enzymes. The addition of micromolar concentrations of cyclic GMP had little effect on the Vmax. for cyclic AMP hydrolysis, but lowered the Km for cyclic AMP hydrolysis to around 20 microM in both cases. However, at low cyclic AMP substrate concentrations, cyclic GMP was a more potent activator of the particulate enzyme than was the soluble enzyme. The activity of these enzymes could be selectively inhibited by cis-16-palmitoleic acid and by arachidonic acid. In each instance, however, the hydrolysis of cyclic AMP became markedly more sensitive to such inhibition when low concentrations of cyclic GMP were present. Tryptic peptide maps of iodinated preparations of these two purified enzyme species showed that there was considerable homology between these two enzyme forms.
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Brooks, S. P. J. "Equilibrium enzymes in metabolic pathways." Biochemistry and Cell Biology 74, no. 3 (May 1, 1996): 411–16. http://dx.doi.org/10.1139/o96-044.
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It is commonly believed that certain reactions in a metabolic sequence may be at or close to equilibrium because of the large excess of catalytic capacity compared to the flux through these enzyme loci. Simple algebraic manipulations can show that the equilibrium and steady state conditions are mutually exclusive. However, solution of the complete reaction schemes for model "equilibrium" reactions shows that they can remain far from equilibrium even though the ratio of enzyme flux to steady state flux through the overall pathway is high. These calculations show that a reaction's proximity to equilibrium depends on the overall flux through the enzyme locus as well as on the kinetic parameters of the other enzymes in the pathway. Thus, combinations of kinetic parameters may exist that allow certain reactions to approach equilibrium but these conditions are not universal.Key words: equilibria, theoretical kinetics, metabolic control.
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Cornish-Bowden, Athel, and Jan-Hendrik S. Hofmeyr. "Enzymes in context: Kinetic characterization of enzymes for systems biology." Biochemist 27, no. 2 (April 1, 2005): 11–14. http://dx.doi.org/10.1042/bio02702011.
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The kinetic behaviour of enzymes is typically observed in conditions appropriate for studying questions of mechanism of action, but these are not necessarily the most appropriate for studying their physiological roles, because they are often too far from those that exist in the living organism. Enzymes therefore need to be studied with natural substrates in the presence of all of the other small molecules likely to affect the activity in vivo, including the reaction products, so that the reverse reaction is not artificially prohibited. As complete reversible rate equations are often unmanageably complicated, especially for cooperative kinetics, care needs to be taken in choosing simpler equations that preserve the properties that are relevant in physiological conditions.
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Raggi, A., and M. Ranieri-Raggi. "Regulatory properties of AMP deaminase isoenzymes from rabbit red muscle." Biochemical Journal 242, no. 3 (March 15, 1987): 875–79. http://dx.doi.org/10.1042/bj2420875.
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We examined the kinetic and regulatory properties of the two isoenzymes of red muscle AMP deaminase, forms A and B, corresponding respectively to the single isoenzymes present in the heart and white skeletal muscle. At the optimal pH value, 6.5, both enzymes show hyperbolic substrate-velocity curves and are inhibited by GTP, inducing sigmoid kinetics. An effect similar to that of GTP is exerted on form B by ATP, whereas form A is almost insensitive to this nucleotide. At pH 7.1 both enzymes follow sigmoid kinetics. ATP enhances the sigmoidicity of the substrate-velocity curve of form B, but it stimulates form A, reverting sigmoidal to hyperbolic kinetics shown by the enzyme at optimal pH. At pH 7.1, form A is also less sensitive to the inhibitory action of Pi and GTP. These results suggest that, owing to the presence of form A, AMP deamination occurs in red muscle also at moderate work intensity. A possible role of this process in counteracting the production of adenosine by 5′-nucleotidase is hypothesized.
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Dong, Jianshu. "On Catalytic Kinetics of Enzymes." Processes 9, no. 2 (January 30, 2021): 271. http://dx.doi.org/10.3390/pr9020271.
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Classical enzyme kinetic theories are summarized and linked with modern discoveries here. The sequential catalytic events along time axis by enzyme are analyzed at the molecular level, and by using master equations, this writing tries to connect the microscopic molecular behavior of enzyme to kinetic data (like velocity and catalytic coefficient k) obtained in experiment: 1/k = t equals to the sum of the times taken by the constituent individual steps. The relationships between catalytic coefficient k, catalytic rate or velocity, the amount of time taken by each step and physical or biochemical conditions of the system are discussed, and the perspective and hypothetic equations proposed here regarding diffusion, conformational change, chemical conversion, product release steps and the whole catalytic cycle provide an interpretation of previous experimental observations and can be testified by future experiments.
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Schnitzer, M. J., and S. M. Block. "Statistical Kinetics of Processive Enzymes." Cold Spring Harbor Symposia on Quantitative Biology 60 (January 1, 1995): 793–802. http://dx.doi.org/10.1101/sqb.1995.060.01.085.
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Ruppe, Alex, Kathryn Mains, and Jerome M. Fox. "A kinetic rationale for functional redundancy in fatty acid biosynthesis." Proceedings of the National Academy of Sciences 117, no. 38 (September 3, 2020): 23557–64. http://dx.doi.org/10.1073/pnas.2013924117.
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Cells build fatty acids with biocatalytic assembly lines in which a subset of enzymes often exhibit overlapping activities (e.g., two enzymes catalyze one or more identical reactions). Although the discrete enzymes that make up fatty acid pathways are well characterized, the importance of catalytic overlap between them is poorly understood. We developed a detailed kinetic model of the fatty acid synthase (FAS) ofEscherichia coliand paired that model with a fully reconstituted in vitro system to examine the capabilities afforded by functional redundancy in fatty acid synthesis. The model captures—and helps explain—the effects of experimental perturbations to FAS systems and provides a powerful tool for guiding experimental investigations of fatty acid assembly. Compositional analyses carried out in silico and in vitro indicate that FASs with multiple partially redundant enzymes enable tighter (i.e., more independent and/or broader range) control of distinct biochemical objectives—the total production, unsaturated fraction, and average length of fatty acids—than FASs with only a single multifunctional version of each enzyme (i.e., one enzyme with the catalytic capabilities of two partially redundant enzymes). Maximal production of unsaturated fatty acids, for example, requires a second dehydratase that is not essential for their synthesis. This work provides a kinetic, control-theoretic rationale for the inclusion of partially redundant enzymes in fatty acid pathways and supplies a valuable framework for carrying out detailed studies of FAS kinetics.
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Ovádi, J., P. Tompa, B. Vértessy, F. Orosz, T. Keleti, and G. R. Welch. "Transient-time analysis of substrate-channelling in interacting enzyme systems." Biochemical Journal 257, no. 1 (January 1, 1989): 187–90. http://dx.doi.org/10.1042/bj2570187.
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The kinetics of dynamically interacting enzyme systems is examined, in the light of increasing evidence attesting to the widespread occurrence of this mode of organization in vivo. The transient time, a key phenomenological parameter for the coupled reaction, is expressed as a function of the lifetime of the intermediate substrate. The relationships between the transient time and the pseudo-first-order rate constants for the coupled reaction by the complexed and uncomplexed enzyme species are indicative of the mechanism of intermediate transfer (‘channelling’). In a dynamically interacting enzyme system these kinetic parameters are composite functions of those for the processes catalysed by the complex and by the separated enzymes. The mathematical paradigm can be extended to a linear sequence of N coupled reactions catalysed by dynamically (pair-wise) interacting enzymes.
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Vannoy, Kathryn J., Andrey Ryabykh, Andrei I. Chapoval, and Jeffrey E. Dick. "Single enzyme electroanalysis." Analyst 146, no. 11 (2021): 3413–21. http://dx.doi.org/10.1039/d1an00230a.
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Traditional enzymology relies on the kinetics of millions of enzymes, an experimental approach that may wash out heterogeneities between individual enzymes. Electrochemical methods have emerged in the last 5 years to probe single enzyme reactivity.
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Duskey, Jason Thomas, Federica da Ros, Ilaria Ottonelli, Barbara Zambelli, Maria Angela Vandelli, Giovanni Tosi, and Barbara Ruozi. "Enzyme Stability in Nanoparticle Preparations Part 1: Bovine Serum Albumin Improves Enzyme Function." Molecules 25, no. 20 (October 9, 2020): 4593. http://dx.doi.org/10.3390/molecules25204593.
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Enzymes have gained attention for their role in numerous disease states, calling for research for their efficient delivery. Loading enzymes into polymeric nanoparticles to improve biodistribution, stability, and targeting in vivo has led the field with promising results, but these enzymes still suffer from a degradation effect during the formulation process that leads to lower kinetics and specific activity leading to a loss of therapeutic potential. Stabilizers, such as bovine serum albumin (BSA), can be beneficial, but the knowledge and understanding of their interaction with enzymes are not fully elucidated. To this end, the interaction of BSA with a model enzyme B-Glu, part of the hydrolase class and linked to Gaucher disease, was analyzed. To quantify the natural interaction of beta-glucosidase (B-Glu,) and BSA in solution, isothermal titration calorimetry (ITC) analysis was performed. Afterwards, polymeric nanoparticles encapsulating these complexes were fully characterized, and the encapsulation efficiency, activity of the encapsulated enzyme, and release kinetics of the enzyme were compared. ITC results showed that a natural binding of 1:1 was seen between B-Glu and BSA. Complex concentrations did not affect nanoparticle characteristics which maintained a size between 250 and 350 nm, but increased loading capacity (from 6% to 30%), enzyme activity, and extended-release kinetics (from less than one day to six days) were observed for particles containing higher B-Glu:BSA ratios. These results highlight the importance of understanding enzyme:stabilizer interactions in various nanoparticle systems to improve not only enzyme activity but also biodistribution and release kinetics for improved therapeutic effects. These results will be critical to fully characterize and compare the effect of stabilizers, such as BSA with other, more relevant therapeutic enzymes for central nervous system (CNS) disease treatments.
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Breger, Joyce, Scott Walper, Mario Ancona, Michael Stewart, Eunkeu Oh, Kimihiro Susumu, and Igor Medintz. "Understanding the Enhanced Kinetics of Enzyme-Quantum Dot Constructs." MRS Advances 1, no. 57 (December 28, 2015): 3831–36. http://dx.doi.org/10.1557/adv.2015.35.
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ABSTRACTBio-inspired, hybrid architectures employing quantum dots (QDs) appended with functionally active biomolecules such as enzymes have the potential to be utilized in numerous applications. Some examples include nanosensors for medical diagnostics, chemical/biological threat detection, as well as “bio-factories” in complex industrial synthetic processes. The main advantage in creating these nanofactories is increased rates in catalysis and efficiency when enzymes are associated with nanoscaffolds, as shown in numerous studies. However, the mechanism for this enhancement remains elusive. Gaining a fundamental, mechanistic understanding of enzyme-QD nanostructures is important in the development of numerous device applications. In this work, we review an array of enzymes attached to QDs and generate a hypothesis in regards to the unique architecture of the enzyme-nanoparticle (NP) construct that leads to increases in catalysis. We highlight work with phosphotiresterase (PTE) attached to two distinctly sized QDs in neutralizing a simulant nerve agent, as well as in other enzyme systems.
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Lloyd, Matthew D. "Steady-state enzyme kinetics." Biochemist 43, no. 3 (May 10, 2021): 40–45. http://dx.doi.org/10.1042/bio_2020_109.
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Steady-state enzyme kinetics is a cornerstone technique of biochemistry and related sciences since it allows the characterization and quantification of enzyme behaviour. Enzyme kinetics is widely used to investigate the physiological role of enzymes, determine the effects of mutations and characterize enzyme inhibitors. Well-known examples of enzyme inhibitors used to treat diseases include anti-infectives (e.g., penicillin, clavulanic acid and HIV protease inhibitors); anti-inflammatories (e.g., aspirin and ibuprofen); cholesterol-lowering statins; tyrosine kinase inhibitors used to treat cancer; and Viagra. Commonly, new disease treatments are discovered by using enzyme kinetics to identify the few active compounds residing within a large compound collection (‘high-throughput screening’). The subject of enzyme kinetics is typically introduced to first-year undergraduates with a mathematical description of behaviour. This Beginners Guide will give a brief overview of experimental enzyme kinetics and the characterization of enzyme inhibitors. Colorimetric assays using a microtitre plate will be considered, although most principles also apply to other assays.
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BECKER, Dieter, Karin S. H. JOHNSON, Anu KOIVULA, Martin SCHÜLEIN, and Michael L. SINNOTT. "Hydrolyses of α- and β-cellobiosyl fluorides by Cel6A (cellobiohydrolase II) of Trichoderma reesei and Humicola insolens." Biochemical Journal 345, no. 2 (January 10, 2000): 315–19. http://dx.doi.org/10.1042/bj3450315.
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We have measured the hydrolyses of α- and β-cellobiosyl fluorides by the Cel6A [cellobiohydrolase II (CBHII)] enzymes of Humicola insolens and Trichoderma reesei, which have essentially identical crystal structures [Varrot, Hastrup, Schülein and Davies (1999) Biochem. J. 337, 297-304]. The β-fluoride is hydrolysed according to Michaelis-Menten kinetics by both enzymes. When the ~ 2.0% of β-fluoride which is an inevitable contaminant in all preparations of the α-fluoride is hydrolysed by Cel7A (CBHI) of T. reesei before initial-rate measurements are made, both Cel6A enzymes show a sigmoidal dependence of rate on substrate concentration, as well as activation by cellobiose. These kinetics are consistent with the classic Hehre resynthesis-hydrolysis mechanism for glycosidase-catalysed hydrolysis of the ‘wrong’ glycosyl fluoride for both enzymes. The Michaelis-Menten kinetics of α-cellobiosyl fluoride hydrolysis by the T. reesei enzyme, and its inhibition by cellobiose, previously reported [Konstantinidis, Marsden and Sinnott (1993) Biochem. J. 291, 883-888] are withdrawn. 1H NMR monitoring of the hydrolysis of α-cellobiosyl fluoride by both enzymes reveals that in neither case is α-cellobiosyl fluoride released into solution in detectable quantities, but instead it appears to be hydrolysed in the enzyme active site as soon as it is formed.
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Meilany, Diah, Efri Mardawati, Made Tri Ari Penia Kresnowati, and Tjandra Setiadi. "KINETIC STUDY OF OIL PALM EMPTY FRUIT BUNCH ENZYMATIC HYDROLYSIS." Reaktor 17, no. 4 (February 2, 2018): 197. http://dx.doi.org/10.14710/reaktor.17.4.197-202.
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As lignocellulosic biomass, Oil Palm Empty Fruit Bunch (OPEFB) can be used as the source of xylose that can be further utilized as the raw material for xylitol production. The processing of OPEFB to xylose comprises of pretreatment and hydrolysis that can be performed enzymatically. This process offers the advantages of moderate operation conditions and more environmentally friendly. This article describes the kinetic study of enzymatic hydrolysis process of OPEFB for producing xylose using self-prepared and commercial xylanase enzymes. Despite the possible mass transfer limitation, the Michaelis Menten kinetics was hypothesized. The results indicated that the reaction at pH 5 and 60°C followed the Michaelis Menten kinetics, with Vm of 0.84 g/L-h and Km of 48.5 g/L for the commercial enzyme, and Vm of 0,38 g/L-h and Km of 0,37 g/L for the self-prepared enzyme. The reaction is affected by temperature, with Ea of 8.6 kcal/gmol. The performance of self-prepared xylanase enzyme was not yet as good as the commercial enzyme, Cellic Htec 2. Keywords: enzymatic hydrolysis; kinetics parameter; OPEFB; xylanase; xylose
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Markin, C. J., D. A. Mokhtari, F. Sunden, M. J. Appel, E. Akiva, S. A. Longwell, C. Sabatti, D. Herschlag, and P. M. Fordyce. "Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics." Science 373, no. 6553 (July 22, 2021): eabf8761. http://dx.doi.org/10.1126/science.abf8761.
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Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a microfluidic platform for high-throughput expression, purification, and characterization of more than 1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium), we performed more than 670,000 reactions and determined more than 5000 kinetic and physical constants for multiple substrates and inhibitors. We uncovered extensive kinetic partitioning to a misfolded state and isolated catalytic effects, revealing spatially contiguous regions of residues linked to particular aspects of function. Regions included active-site proximal residues but extended to the enzyme surface, providing a map of underlying architecture not possible to derive from existing approaches. HT-MEK has applications that range from understanding molecular mechanisms to medicine, engineering, and design.
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MARCEL, Véronique, Laurent Gagnoux PALACIOS, Christophe PERTUY, Patrick MASSON, and Didier FOURNIER. "Two invertebrate acetylcholinesterases show activation followed by inhibition with substrate concentration." Biochemical Journal 329, no. 2 (January 15, 1998): 329–34. http://dx.doi.org/10.1042/bj3290329.
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In vertebrates there are two cholinesterases, with differences in catalytic behaviour with respect to substrate concentration: butyrylcholinesterase displays an increased activity at low substrate concentrations, whereas acetylcholinesterase displays inhibition by excess substrate. In two invertebrates, Drosophila melanogaster and Caenorhabditis elegans, we found cholinesterases that showed both kinetic complexities: substrate activation at low substrate concentrations followed by inhibition at higher concentrations. These triphasic kinetics can be explained by the presence of two enzymes with different kinetic behaviours or more probably by the existence of a single enzyme regulated by the substrate concentration.
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Chisti, Yusuf. "Interfacial enzymes: a neglected domain of biocatalysis. Interfacial Enzyme Kinetics." Biotechnology Advances 20, no. 3-4 (November 2002): 269–70. http://dx.doi.org/10.1016/s0734-9750(02)00014-9.
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Fink, A. M. "Optimal control in liver kinetics." Journal of the Australian Mathematical Society. Series B. Applied Mathematics 27, no. 3 (January 1986): 361–69. http://dx.doi.org/10.1017/s0334270000004987.
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AbstractWe solve a minimization problem in liver kinetics posed by Bass, et al., in this journal, (1984), pages 538–562. The problem is to choose the density functions for the location of two enzymes, in order to minimize the concentration of an intermediate form of a substance at the outlet of the liver. This form may be toxic to the rest of the body, but the second enzyme renders it harmless. It seems natural that the second enzyme should be downstream from the first. However, we can show that the minimum problem is sometimes solved by an overlap of the supports of the two density functions. Even more surprising is that, for certain forms of the kinetic functions and high levels of transformation of the first enzymatic reaction, some of the first enzyme should be located downstream from all the second enzyme. This suggests that the first reaction should be relatively slow.
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Runge, Steven W., Brent J. F. Hill, and William M. Moran. "A Simple Classroom Teaching Technique To Help Students Understand Michaelis-Menten Kinetics." CBE—Life Sciences Education 5, no. 4 (December 2006): 348–52. http://dx.doi.org/10.1187/cbe.06-04-0160.
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A new, simple classroom technique helps cell biology students understand principles of Michaelis-Menten enzyme kinetics. A student mimics the enzyme and the student's hand represents the enzyme's active site. The catalytic event is the transfer of marbles (substrate molecules) by hand from one plastic container to another. As predicted, increases in marble concentration increase the number of marbles transferred per unit time (initial rate, V0) until the turnover number becomes rate limiting and V0 approaches the maximum velocity (Vmax), as described by the Michaelis-Menten equation. With this demonstration, students visualize an important concept: the turnover number is constant and independent of marble concentration. A student assessment of this exercise showed that it helped students visualize the turnover number and Vmax but not Km, the marble concentration at which V0 is one-half Vmax. To address the concept of Km, we use supplemental laboratory and lecture exercises. This exercise with plastic containers and marbles is equally suited to demonstrate the kinetics of carrier-mediated membrane transport. We conclude that this exercise helps students visualize the turnover number and Vmax and gives students important insights into the kinetic parameters used to characterize the catalytic activity of enzymes and membrane transporters.
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Martín, J., J. Pérez-Gil, C. Acebal, and R. Arche. "Theoretical approach to the steady-state kinetics of a bi-substrate acyl-transfer enzyme reaction that follows a hydrolysable-acyl-enzyme-based mechanism. Application to the study of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase from rabbit lung." Biochemical Journal 266, no. 1 (February 15, 1990): 47–53. http://dx.doi.org/10.1042/bj2660047.
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A kinetic model is proposed for catalysis by an enzyme that has several special characteristics: (i) it catalyses an acyl-transfer bi-substrate reaction between two identical molecules of substrate, (ii) the substrate is an amphiphilic molecule that can be present in two physical forms, namely monomers and micelles, and (iii) the reaction progresses through an acyl-enzyme-based mechanism and the covalent intermediate can react also with water to yield a secondary hydrolytic reaction. The theoretical kinetic equations for both reactions were deduced according to steady-state assumptions and the theoretical plots were predicted. The experimental kinetics of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase from rabbit lung fitted the proposed equations with great accuracy. Also, kinetics of inhibition by products behaved as expected. It was concluded that the competition between two nucleophiles for the covalent acyl-enzyme intermediate, and not a different enzyme action depending on the physical state of the substrate, is responsible for the differences in kinetic pattern for the two activities of the enzyme. This conclusion, together with the fact that the kinetic equation for the transacylation is quadratic, generates a ‘hysteretic’ pattern that can provide the basis of self-regulatory properties for enzymes to which this model could be applied.
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Chen, Yu, Feiran Li, Jiwei Mao, Yun Chen, and Jens Nielsen. "Yeast optimizes metal utilization based on metabolic network and enzyme kinetics." Proceedings of the National Academy of Sciences 118, no. 12 (March 15, 2021): e2020154118. http://dx.doi.org/10.1073/pnas.2020154118.
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Metal ions are vital to metabolism, as they can act as cofactors on enzymes and thus modulate individual enzymatic reactions. Although many enzymes have been reported to interact with metal ions, the quantitative relationships between metal ions and metabolism are lacking. Here, we reconstructed a genome-scale metabolic model of the yeast Saccharomyces cerevisiae to account for proteome constraints and enzyme cofactors such as metal ions, named CofactorYeast. The model is able to estimate abundances of metal ions binding on enzymes in cells under various conditions, which are comparable to measured metal ion contents in biomass. In addition, the model predicts distinct metabolic flux distributions in response to reduced levels of various metal ions in the medium. Specifically, the model reproduces changes upon iron deficiency in metabolic and gene expression levels, which could be interpreted by optimization principles (i.e., yeast optimizes iron utilization based on metabolic network and enzyme kinetics rather than preferentially targeting iron to specific enzymes or pathways). At last, we show the potential of using the model for understanding cell factories that harbor heterologous iron-containing enzymes to synthesize high-value compounds such as p-coumaric acid. Overall, the model demonstrates the dependence of enzymes on metal ions and links metal ions to metabolism on a genome scale.
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Rojas, Bruno E., Matías D. Hartman, Carlos M. Figueroa, Laura Leaden, Florencio E. Podestá, and Alberto A. Iglesias. "Biochemical characterization of phosphoenolpyruvate carboxykinases from Arabidopsis thaliana." Biochemical Journal 476, no. 20 (October 18, 2019): 2939–52. http://dx.doi.org/10.1042/bcj20190523.
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Abstract ATP-dependent phosphoenolpyruvate carboxykinases (PEPCKs, EC 4.1.1.49) from C4 and CAM plants have been widely studied due to their crucial role in photosynthetic CO2 fixation. However, our knowledge on the structural, kinetic and regulatory properties of the enzymes from C3 species is still limited. In this work, we report the recombinant production and biochemical characterization of two PEPCKs identified in Arabidopsis thaliana: AthPEPCK1 and AthPEPCK2. We found that both enzymes exhibited high affinity for oxaloacetate and ATP, reinforcing their role as decarboxylases. We employed a high-throughput screening for putative allosteric regulators using differential scanning fluorometry and confirmed their effect on enzyme activity by performing enzyme kinetics. AthPEPCK1 and AthPEPCK2 are allosterically modulated by key intermediates of plant metabolism, namely succinate, fumarate, citrate and α-ketoglutarate. Interestingly, malate activated and glucose 6-phosphate inhibited AthPEPCK1 but had no effect on AthPEPCK2. Overall, our results demonstrate that the enzymes involved in the critical metabolic node constituted by phosphoenolpyruvate are targets of fine allosteric regulation.
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Küchler, Jan, Katharina Willenbücher, Elisabeth Reiß, Lea Nuß, Marius Conrady, Patrice Ramm, Ulrike Schimpf, Udo Reichl, Ulrich Szewzyk, and Dirk Benndorf. "Degradation Kinetics of Lignocellulolytic Enzymes in a Biogas Reactor Using Quantitative Mass Spectrometry." Fermentation 9, no. 1 (January 12, 2023): 67. http://dx.doi.org/10.3390/fermentation9010067.
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The supplementation of lignocellulose-degrading enzymes can be used to enhance the performance of biogas production in industrial biogas plants. Since the structural stability of these enzyme preparations is essential for efficient application, reliable methods for the assessment of enzyme stability are crucial. Here, a mass-spectrometric-based assay was established to monitor the structural stability of enzymes, i.e., the structural integrity of these proteins, in anaerobic digestion (AD). The analysis of extracts of Lentinula edodes revealed the rapid degradation of lignocellulose-degrading enzymes, with an approximate half-life of 1.5 h. The observed low structural stability of lignocellulose-degrading enzymes in AD corresponded with previous results obtained for biogas content. The established workflow can be easily adapted for the monitoring of other enzyme formulations and provides a platform for evaluating the effects of enzyme additions in AD, together with a characterization of the biochemical methane potential used in order to determine the biodegradability of organic substrates.
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Labourel, Florian, and Etienne Rajon. "Resource Uptake and the Evolution of Moderately Efficient Enzymes." Molecular Biology and Evolution 38, no. 9 (May 8, 2021): 3938–52. http://dx.doi.org/10.1093/molbev/msab132.
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Abstract Enzymes speed up reactions that would otherwise be too slow to sustain the metabolism of selfreplicators. Yet, most enzymes seem only moderately efficient, exhibiting kinetic parameters orders of magnitude lower than their expected physically achievable maxima and spanning over surprisingly large ranges of values. Here, we question how these parameters evolve using a mechanistic model where enzyme efficiency is a key component of individual competition for resources. We show that kinetic parameters are under strong directional selection only up to a point, above which enzymes appear to evolve under near-neutrality, thereby confirming the qualitative observation of other modeling approaches. While the existence of a large fitness plateau could potentially explain the extensive variation in enzyme features reported, we show using a population genetics model that such a widespread distribution is an unlikely outcome of evolution on a common landscape, as mutation–selection–drift balance occupy a narrow area even when very moderate biases towards lower efficiency are considered. Instead, differences in the evolutionary context encountered by each enzyme should be involved, such that each evolves on an individual, unique landscape. Our results point to drift and effective population size playing an important role, along with the kinetics of nutrient transporters, the tolerance to high concentrations of intermediate metabolites, and the reversibility of reactions. Enzyme concentration also shapes selection on kinetic parameters, but we show that the joint evolution of concentration and efficiency does not yield extensive variance in evolutionary outcomes when documented costs to protein expression are applied.
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Zdražilová, Pavla, Šárka Štĕpánková, Alena Komersová, Martina Vránová, Karel Komers, and Alexander Čegan. "Kinetics of 13 New Cholinesterase Inhibitors." Zeitschrift für Naturforschung C 61, no. 7-8 (August 1, 2006): 611–17. http://dx.doi.org/10.1515/znc-2006-7-823.
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Kinetics of hydrolysis of acetylcholine and acetylthiocholine by two types of acetylcholinesterase and butyrylcholinesterase inhibited by 13 new inhibitors (5 carbamates and 8 carbazates - hydrazinium derivatives) was measured in vitro in a batch reactor at 25 °C, pH 8, ionic strength 0.11 ᴍ and enzyme activity 3.5 U by four nondependent analytical methods. Sevin®, rivastigmin (Exelon®) and galantamin (Reminyl®) served as comparative inhibiting standards. Kinetics of hydrolyses inhibited by all studied carbamates, sevin, carbazates (with exceptions) and rivastigmin (with exceptions) can be simulated by the competitive inhibition model with irreversible reaction between enzyme and inhibitor. Galantamin does not fulfil this model. In positive simulations, the value of inhibition (carbamoylation) rate constant k3 was calculated, describing the reaction velocity between the given enzyme and inhibitor. Physiologically important hydrolyses of acetylcholine catalyzed by acetylcholinesterase from electric eel or bovine erythrocytes and butyrylcholinesterase from horse plasma can be most quickly inhibited by carbamoylation of the mentioned enzymes by the 3-N,N-diethylaminophenyl- N′-(1-alkyl) carbamates 4 and 5. Probably this is due to a long enough hydrocarbon aliphatic substituent (hexyl and octyl) on the amidic nitrogen atom. The tested carbazates failed as inhibitors of cholinesterases. The regeneration ability of the inhibited enzymes was not measured.
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Vlad-Oros, B., G. Preda, Z. Dudas, M. Dragomirescu, and A. Chiriac. "Entrapment of glucoamylase by sol-gel technique in PhTES/TEOS hybrid matrixes." Processing and Application of Ceramics 1, no. 1-2 (2007): 63–67. http://dx.doi.org/10.2298/pac0702063v.
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Mesoporous silica particles were prepared by the sol-gel method from different alkoxysilane precursors and used as a host matrix for encapsulation of glucoamylase, an enzyme widely used in fermentative industry. The aim was to investigate the physico-chemical properties of the different silica powders and their effect on the enzyme kinetics. The encapsulated enzymes followed Michaelis-Menten kinetics. The Michaelis constant (KM) and the maximum rate of starch hydrolysis reaction (Vmax) were calculated according to the Michaelis-Menten and Lineweaver-Burke plots. The values of the Michaelis constant (KM) of the encapsulated enzymes were higher than those of the free enzyme. The temperature and pH influence on the activity of free and immobilized glucoamylase were also compared. The results of this study show that the enzymes immobilized in organic/inorganic hybrid silica matrixes (obtained by the sol-gel method), allowing the entrapped glucoamylase to retain its biological activity, are suitable for many different applications, (medicinal, clinical, analytical).
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DE ATAURI, Pedro, Luis ACERENZA, Boris N. KHOLODENKO, Núria DE LA IGLESIA, Joan J. GUINOVART, Loranne AGIUS, and Marta CASCANTE. "Occurrence of paradoxical or sustained control by an enzyme when overexpressed: necessary conditions and experimental evidence with regard to hepatic glucokinase." Biochemical Journal 355, no. 3 (April 24, 2001): 787–93. http://dx.doi.org/10.1042/bj3550787.
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It is widely assumed that the control coefficient of an enzyme on pathway flux decreases as the concentration of enzyme increases. However, it has been shown [Kholodenko and Brown (1996) Biochem. J. 314, 753–760] that enzymes with sigmoidal kinetics can maintain or even gain control with an increase in enzyme activity or concentration. This has been described as ‘paradoxical control’. Here we formulate the general requirements for allosteric enzyme kinetics to display this behaviour. We show that a necessary condition is that the Hill coefficient of the enzyme should increase with an increase in substrate concentration or decrease with an increase in product concentration. We also describe the necessary and sufficient requirements for the occurrence of paradoxical control in terms of the flux control coefficients and the derivatives of the elasticities. The derived expression shows that the higher the control coefficient of an allosteric enzyme, the more likely it is that the pathway will display this behaviour. Control of pathway flux is generally shared between a large number of enzymes and therefore the likelihood of observing sustained or increased control is low, even if the kinetic parameters are in the most favourable range to generate the phenomenon. We show that hepatic glucokinase, which has a very high flux control coefficient and displays sigmoidal behaviour within the hepatocyte in situ as a result of interaction with a regulatory protein, displays sustained or increased control over an extended range of enzyme concentrations when the regulatory protein is overexpressed.
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Becker, Erwin W. "Dynamics and Kinetics of Enzymes Kinetic Equilibrium of Forces in Biochemistry." Zeitschrift für Naturforschung C 47, no. 7-8 (August 1, 1992): 628–33. http://dx.doi.org/10.1515/znc-1992-7-823.
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To explain the high specificity, high reaction rate, and high thermodynamic efficiency in enzymatic processes, cooperation of the enzyme with a molecular transfer unit is assumed. A “kinetic equilibrium of forces” is suggested, which enables high reaction rates to occur under equilibrium conditions and a thorough examination of the substrate to be made without consumption of free energy. In case of ATPases, ion-binding proteins are the most probable transfer units. By analyzing the elementary effect in muscle contraction it is shown that the new theorem may be of substantial value in elucidating biochemical processes.
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Barroso, Juan B., Juan Peragón, Constanza Contreras-Jurado, Leticia García-Salguero, Francisco J. Corpas, Francisco J. Esteban, María A. Peinado, Manuel De La Higuera, and José A. Lupiáñez. "Impact of starvation-refeeding on kinetics and protein expression of trout liver NADPH-production systems." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 274, no. 6 (June 1, 1998): R1578—R1587. http://dx.doi.org/10.1152/ajpregu.1998.274.6.r1578.
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Herein we report on the kinetic and protein expression of glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase, and malic enzyme (ME) in the liver of the trout ( Oncorhynchus mykiss) during a long-term starvation-refeeding cycle. Starvation significantly depressed the activity of these enzymes by almost 60%, without changing the Michaelis constant. The time response to this nutritional stimulus increased with fish weight. The sharp decline in G6PDH and ME activities was due to a specific protein-repression phenomenon, as demonstrated by molecular and immunohistochemical analyses. Also, the dimeric banding pattern of liver G6PDH shifted from the fully reduced and partially oxidized forms, predominant in control, to a fully oxidized form, more sensitive to proteolytic inactivation. Refeeding caused opposite effects in both protein concentration and enzyme activities of about twice the control values in the first stages, later reaching the normal enzyme activity levels. Additionally, the partially oxidized form of G6PDH increased. The kinetics of these enzymes were examined in relation to the various metabolic roles of NADPH. These results clearly indicate that trout liver undergoes protein repression-induction processes under these two contrasting nutritional conditions.
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Valchev, Ivo, Nikolay Yavorov, and Stoyko Petrin. "Topochemical kinetic mechanism of cellulase hydrolysis on fast-growing tree species. COST Action FP1105." Holzforschung 70, no. 12 (December 1, 2016): 1147–53. http://dx.doi.org/10.1515/hf-2016-0030.
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Abstract Optimization of the lignocellulosic bioconversion by cellulolytic enzymes requires good knowledge of reaction kinetics. In the present paper, the investigations of the kinetics have been performed on the fast-growing tree species of poplar, paulownia, willow and black locust, which were pretreated by steam explosion (SE), and bleached kraft pulp (BKP) made of a hardwood mixture. The applicability of different kinetic equations referring to diffusion, topochemical and other heterogeneous catalytic processes was examined, and it was found that the enzyme process is best described by the modified Prout-Tompkins topochemical equation. According to that kinetic model, the hydrolysis rate depends on the amount of the substrate left and the inhibition of the enzyme by the product formed and, moreover, on the combination of chemical interaction and diffusion processes. There is a compensation effect between activation energy and pre-exponential factor and there are correlations between rate constant, power factor, and wood density. The mechanisms of cellulase hydrolysis of BKP- and SE-treated fast-growing tree species are very similar. The results shows that the structural features of the lignocellulosic material are the controlling factor on the type of the kinetic mechanism. The obtained temperature-time dependence of degree of enzyme hydrolysis is useful for simulation and control of the process.
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Craig, Jonathan M., Andrew H. Laszlo, Henry Brinkerhoff, Ian M. Derrington, Matthew T. Noakes, Ian C. Nova, Benjamin I. Tickman, Kenji Doering, Noah F. de Leeuw, and Jens H. Gundlach. "Revealing dynamics of helicase translocation on single-stranded DNA using high-resolution nanopore tweezers." Proceedings of the National Academy of Sciences 114, no. 45 (October 16, 2017): 11932–37. http://dx.doi.org/10.1073/pnas.1711282114.
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Enzymes that operate on DNA or RNA perform the core functions of replication and expression in all of biology. To gain high-resolution access to the detailed mechanistic behavior of these enzymes, we developed single-molecule picometer-resolution nanopore tweezers (SPRNT), a single-molecule technique in which the motion of polynucleotides through an enzyme is measured by a nanopore. SPRNT reveals two mechanical substates of the ATP hydrolysis cycle of the superfamily 2 helicase Hel308 during translocation on single-stranded DNA (ssDNA). By analyzing these substates at millisecond resolution, we derive a detailed kinetic model for Hel308 translocation along ssDNA that sheds light on how superfamily 1 and 2 helicases turn ATP hydrolysis into motion along DNA. Surprisingly, we find that the DNA sequence within Hel308 affects the kinetics of helicase translocation.
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Goel, Rajeev, Takashi Mino, Hiroyasu Satoh, and Tomonori Matsuo. "Comparison of hydrolytic enzyme systems in pure culture and activated sludge under different electron acceptor conditions." Water Science and Technology 37, no. 4-5 (February 1, 1998): 335–43. http://dx.doi.org/10.2166/wst.1998.0659.
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Enzymatic hydrolysis under different electron acceptor conditions in nutrient removal activated sludge treatment processes is a weak link in the Activated Sludge Model no. 2 (Henze et al., 1995). An experimental study was undertaken to gain insight into the hydrolysis process with specific focus on hydrolysis kinetics and rates under different electron acceptor conditions. Two pure cultures, Bacillus amyloliquefaciens (Gram positive) and Pseudomonas saccharophila (Gram negative) were chosen for the study. In addition, activated sludge grown in an anaerobic-aerobic system was tested for enzymatic activity using starch as the model substrate. The hydrolytic enzymes were found to be released into the bulk in pure cultures whereas the enzyme activity was found to be mainly associated with the cell surfaces in activated sludge. Further, it was observed that the development of the hydrolytic enzyme system in Bacillus amyloliquefaciens and P. saccharophila is strongly suppressed under anoxic and anaerobic conditions. However, the effect of anaerobic and aerobic incubation on hydrolytic enzyme activity in activated sludge was found to be small. Starch hydrolysis kinetic data from batch experiments with activated sludge followed substrate saturation kinetics that were linear with biomass concentration. Finally, the similar hydrolytic enzyme activities observed under anaerobic and aerobic phases of a sequencing batch reactor are explained by considering the aspects of enzyme location and enzyme system development under aerobic and anaerobic phases. It is proposed that the floc bound enzymes are recycled in a single sludge system so that an equilibrium exists between enzyme loss and synthesis at steady state.
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Ford, Jonathan B., Mac T. Wayment, Timothy E. Albertson, Kelly P. Owen, Joshua B. Radke, and Mark E. Sutter. "Elimination Kinetics of Ethanol in a 5-Week-Old Infant and a Literature Review of Infant Ethanol Pharmacokinetics." Case Reports in Medicine 2013 (2013): 1–3. http://dx.doi.org/10.1155/2013/250716.
Full textAbstract:
Primary ethanol metabolism occurs through alcohol dehydrogenase, but minor metabolic pathways such as the P450 enzymes CYP2E1 and CYP1A2 and the enzyme catalase exist. These enzymes have distinct developmental stages. Elimination kinetics of ethanol in the infant is limited. We report the elimination kinetics of ethanol in a 5-week-old African-American male who had a serum ethanol level of 270 mg/dL on admission. A previously healthy 5-week-old African-American male was brought to the ED with a decreased level of consciousness. His initial blood ethanol level was 270 mg/dL. Serial blood ethanol levels were obtained. The elimination rate of ethanol was calculated to be in a range from 17.1 to 21.2 mg/dL/hr and appeared to follow zero-order elimination kinetics with aR2=0.9787. Elimination kinetics for ethanol in the young infant has been reported in only four previously published reports. After reviewing these reports, there appears to be variability in the elimination rates of ethanol in infants. Very young infants may not eliminate ethanol as quickly as previously described. Given that there are different stages of enzyme development in children, caution should be used when generalizing the elimination kinetics in young infants and children.
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Page, M. G. P. "The kinetics of non-stoichiometric bursts of β-lactam hydrolysis catalysed by class C β-lactamases." Biochemical Journal 295, no. 1 (October 1, 1993): 295–304. http://dx.doi.org/10.1042/bj2950295.
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Class C beta-lactamases from Pseudomonas aeruginosa and several species of the Enterobacteriaceae have been observed to undergo a rapid burst in hydrolysis of beta-lactam antibiotics before relaxation to a steady-state rate of hydrolysis. The amplitude of the burst corresponds to the hydrolysis of between 1 and 10,000 mol of the substrate per mol of enzyme. The decay of the rate of hydrolysis in the burst phase comprises two exponential reactions, which indicates that there are three different reactive states of the enzymes. Examination of the kinetics of acylation by slowly reacting beta-lactams suggests that there are three forms of the free enzyme in slow equilibrium. Thus it would appear that the burst kinetics exhibited by class C enzymes can be attributed to redistribution of the enzyme between different conformations induced by the reaction with substrate.
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Gallardo Aguilar, Irenia, Yedier Rodríguez Padrón, Yisel Pérez Milian, Fernando Sarría Quesada, and Juan P. Hernández Touset. "Kinetics of the hydrolysis of sorghum flour with two amilaceouos enzymes." Afinidad. Journal of Chemical Engineering Theoretical and Applied Chemistry 80, no. 598 (March 30, 2023): 94–98. http://dx.doi.org/10.55815/413459.
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In the production process of glucose syrup, the conversion of corn starches to sugars occurs through acid or enzymatic hydrolysis. Corn has been the traditional raw material in its preparation. The use of other cereals, such as sorghum, is reported for its similar properties to corn. The objective of the work is to study the kinetic behavior in the hydrolysis of sorghum flour, using two enzymes: Bialfa-T and Glucozyme 2X in the production of syrup. The sorghum used was UDG 110. Two experimental screening designs 22 to study the effect of enzymes on sorghum flour were performed. The variables studied were: substrate concentration and enzyme concentration and as response variables reducing sugars (RS) (g/L), conversion (%) and enzymatic activity (UI). The regression models for the three response variables were significant for the Bialfa-T enzyme, while for Glucozyme 2X, only the enzymatic activity model was significant, since this enzyme acts on already transformed starches and the variable with the highest influence turned out to be the substrate concentration, followed by the enzyme concentration.
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Candy, J. M., and R. G. Duggleby. "Investigation of the cofactor-binding site of Zymomonas mobilis pyruvate decarboxylase by site-directed mutagenesis." Biochemical Journal 300, no. 1 (May 15, 1994): 7–13. http://dx.doi.org/10.1042/bj3000007.
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Several enzymes require thiamin diphosphate (ThDP) as an essential cofactor, and we have used one of these, pyruvate decarboxylase (PDC; EC 4.1.1.1) from Zymomonas mobilis, as a model for this group of enzymes. It is well suited for this purpose because of its stability, ease of purification and its simple kinetic properties. A sequence motif of approx. 30 residues, beginning with a glycine-aspartate-glycine (-GDG-) triplet and ending with a double asparagine (-NN-) sequence, has been identified in many of these enzymes [Hawkins, Borges and Perham (1989) FEBS Lett. 255, 77-82]. Other residues within this putative ThDP-binding motif are conserved, but to a lesser extent, including a glutamate and a proline residue. The role of the elements of this motif has been clarified by the determination of the three-dimensional structure of three of these enzymes [Muller, Lindqvist, Furey, Schulz, Jordan and Schneider (1993) Structure 1, 95-103]. Four of the residues within this motif were modified by site-directed mutagenesis of the cloned PDC gene to evaluate their role in cofactor binding. The mutant proteins were expressed in Escherichia coli and found to purify normally, indicating that the tertiary structure of these enzymes had not been grossly perturbed by the amino acid substitutions. We have shown previously [Diefenbach, Candy, Mattick and Duggleby (1992) FEBS Lett. 296, 95-98] that changing the aspartate in the -GDG- sequence to glycine, threonine or asparagine yields an inactive enzyme that is unable to bind ThDP, therefore verifying the role of the ThDP-binding motif. Here we demonstrate that substitution with glutamate yields an active enzyme with a greatly reduced affinity for both ThDP and Mg2+, but with normal kinetics for pyruvate. Unlike the wild-type tetrameric enzyme, this mutant protein usually exists as a dimer. Replacement of the second asparagine of the -NN- sequence by glutamine also yields an inactive enzyme which is unable to bind ThDP, whereas replacement with an aspartate residue results in an active enzyme with a reduced affinity for ThDP but which displays normal kinetics for both Mg2+ and pyruvate. Replacing the conserved glutamate with aspartate did not alter the properties of the enzyme, while the conserved proline, thought to be required for structural reasons, could be substituted with glycine or alanine without inactivating the enzyme, but these changes did reduce its stability.
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Tang, J. Y. "On the relationships between Michaelis–Menten kinetics, reverse Michaelis–Menten kinetics, Equilibrium Chemistry Approximation kinetics and quadratic kinetics." Geoscientific Model Development Discussions 8, no. 9 (September 3, 2015): 7663–91. http://dx.doi.org/10.5194/gmdd-8-7663-2015.
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Abstract. The Michaelis–Menten kinetics and the reverse Michaelis–Menten kinetics are two popular mathematical formulations used in many land biogeochemical models to describe how microbes and plants would respond to changes in substrate abundance. However, the criteria of when to use which of the two are often ambiguous. Here I show that these two kinetics are special approximations to the Equilibrium Chemistry Approximation kinetics, which is the first order approximation to the quadratic kinetics that solves the equation of enzyme-substrate complex exactly for a single enzyme single substrate biogeochemical reaction with the law of mass action and the assumption of quasi-steady-state for the enzyme-substrate complex and that the product genesis from enzyme-substrate complex is much slower than the equilibration between enzyme-substrate complexes, substrates and enzymes. In particular, I showed that the derivation of the Michaelis–Menten kinetics does not consider the mass balance constraint of the substrate, and the reverse Michaelis–Menten kinetics does not consider the mass balance constraint of the enzyme, whereas both of these constraints are taken into account in the Equilibrium Chemistry Approximation kinetics. By benchmarking against predictions from the quadratic kinetics for a wide range of substrate and enzyme concentrations, the Michaelis–Menten kinetics was found to persistently under-predict the normalized sensitivity ∂ ln v / ∂ ln k2+ of the reaction velocity v with respect to the maximum product genesis rate k2+, persistently over-predict the normalized sensitivity ∂ ln v / ∂ ln k1+ of v with respect to the intrinsic substrate affinity k1+, persistently over-predict the normalized sensitivity ∂ ln v / ∂ ln [ E ]T of v with respect the total enzyme concentration [ E ]T and persistently under-predict the normalized sensitivity ∂ ln v / ∂ ln [ S ]T of v with respect to the total substrate concentration [ S ]T. Meanwhile, the reverse Michaelis–Menten kinetics persistently under-predicts ∂ ln v / ∂ ln k2+ and ∂ ln v / ∂ ln [ E ]T, and persistently over-predicts ∂ ln v / ∂ ln k1+ and ∂ ln v / ∂ ln [ S ]T. In contrast, the Equilibrium Chemistry Approximation kinetics always gives consistent predictions of ∂ ln v / ∂ ln k2+, ∂ ln v / ∂ ln k1+, ∂ ln v / ∂ ln [ E ]T and ∂ ln v / ∂ ln [ S ]T. Since the Equilibrium Chemistry Approximation kinetics includes the advantages from both the Michaelis–Menten kinetics and the reverse Michaelis–Menten kinetics and it is applicable for almost the whole range of substrate and enzyme abundances, soil biogeochemical modelers therefore no longer need to choose when to use the Michaelis–Menten kinetics or the reverse Michaelis–Menten kinetics. I expect removing this choice ambiguity will make it easier to formulate more robust and consistent land biogeochemical models.
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Høst, Amalie Vang, Roberto Morellon-Sterling, Diego Carballares, John M. Woodley, and Roberto Fernandez-Lafuente. "Co-Enzymes with Dissimilar Stabilities: A Discussion of the Likely Biocatalyst Performance Problems and Some Potential Solutions." Catalysts 12, no. 12 (December 3, 2022): 1570. http://dx.doi.org/10.3390/catal12121570.
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Enzymes have several excellent catalytic features, and the last few years have seen a revolution in biocatalysis, which has grown from using one enzyme to using multiple enzymes in cascade reactions, where the product of one enzyme reaction is the substrate for the subsequent one. However, enzyme stability remains an issue despite the many benefits of using enzymes in a catalytic system. When enzymes are exposed to harsh process conditions, deactivation occurs, which changes the activity of the enzyme, leading to an increase in reaction time to achieve a given conversion. Immobilization is a well-known strategy to improve many enzyme properties, if the immobilization is properly designed and controlled. Enzyme co-immobilization is a further step in the complexity of preparing a biocatalyst, whereby two or more enzymes are immobilized on the same particle or support. One crucial problem when designing and using co-immobilized enzymes is the possibility of using enzymes with very different stabilities. This paper discusses different scenarios using two co-immobilized enzymes of the same or differing stability. The effect on operational performance is shown via simple simulations using Michaelis–Menten equations to describe kinetics integrated with a deactivation term. Finally, some strategies for overcoming some of these problems are discussed.
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Liu, Yan, Yali Shen, Yang Song, Lei Xu, J. Jefferson P. P. Perry, and Jiayu Liao. "Isopeptidase Kinetics Determination by a Real Time and Sensitive qFRET Approach." Biomolecules 11, no. 5 (April 30, 2021): 673. http://dx.doi.org/10.3390/biom11050673.
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Isopeptidase activity of proteases plays critical roles in physiological and pathological processes in living organisms, such as protein stability in cancers and protein activity in infectious diseases. However, the kinetics of protease isopeptidase activity has not been explored before due to a lack of methodology. Here, we report the development of novel qFRET-based protease assay for characterizing the isopeptidase kinetics of SENP1. The reversible process of SUMOylation in vivo requires an enzymatic cascade that includes E1, E2, and E3 enzymes and Sentrin/SUMO-specific proteases (SENPs), which can act either as endopeptidases that process the pre-SUMO before its conjugation, or as isopeptidases to deconjugate SUMO from its target substrate. We first produced the isopeptidase substrate of CyPet-SUMO1/YPet-RanGAP1c by SUMOylation reaction in the presence of SUMO E1 and E2 enzymes. Then a qFRET analyses of real-time FRET signal reduction of the conjugated substrate of CyPet-SUMO1/YPet-RanGAP1c to free CyPet-SUMO1 and YPet-RanGAP1c by the SENP1 were able to obtain the kinetic parameters, Kcat, KM, and catalytic efficiency (Kcat/KM) of SENP1. This represents a pioneer effort in isopeptidase kinetics determination. Importantly, the general methodology of qFRET-based protease isopeptidase kinetic determination can also be applied to other proteases.
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Rangan, A., and V. Arunachalam. "A Stochastic Model for Cell Repair Based on Enzyme Kinetics." Journal of Biological Systems 05, no. 01 (March 1997): 139–50. http://dx.doi.org/10.1142/s0218339097000114.
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A new stochastic model for the repair of radiation-induced cell damage based on enzyme kinetics is proposed. The model not only accounts for the depletion of enzyme level with successive repairs but also takes into account the synthesis of new enzymes in response to repairs. The cell survival probability is explicitly calculated, using which comparisons with related models are made.
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Megawati, B. Triwibowo, Z. A. S. Bahlawan, Z. Fitriani, and N. Ulfah. "Kinetic study on hydrolysis of sweet sorghum (Sorghum bicolor (L.) Moench) stem dregs with cocktail enzymes in bioethanol production." IOP Conference Series: Earth and Environmental Science 1203, no. 1 (June 1, 2023): 012005. http://dx.doi.org/10.1088/1755-1315/1203/1/012005.
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Abstract Sweet sorghum stem dregs have potential as an alternative raw material for bioethanol production due to its high sugar content. The purpose of this study is to examine the hydrolysis kinetics of sweet sorghum stem dregs with cocktail enzymes using several models, namely the Valjamae, Kopelman, and Gompertz models. The hydrolysis was carried out at a concentration of 1/16.7 g/mL, a temperature of 50°C, and at variations of time (0, 12, 24, 36, 48, 60 and 72 minutes). The cocktail enzyme used was Multifect CL, which contains endoglucanases, exoglucanases, and β-glucosidases and has the potential to convert lignocellulose. The reactor used is CSTR (Continuous Stirred-Tank Reactor). The study results indicated that the hydrolysis kinetics of the Valjamae and Kopelman models were very suitable. The value of reaction rate constant and exponential fractal for the Valjamae model are 0.077 L/hour and 0.434 and for the Kopelman model are 0.044 L/hour and 0.415. The hydrolysis kinetics for the Gompertz model is not suitable. These hydrolysis kinetic parameters can be utilized in the bioreactors planning that will be used in the preliminary design of the biomass-based bioethanol industry.
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Stuchebrukhov, Alexei A. "Kinetics and Efficiency of Energy-Transducing Enzymes." Journal of Physical Chemistry B 123, no. 44 (September 26, 2019): 9456–65. http://dx.doi.org/10.1021/acs.jpcb.9b08191.
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Park, Seong Jun, Sanggeun Song, In-Chun Jeong, Hye Ran Koh, Ji-Hyun Kim, and Jaeyoung Sung. "Nonclassical Kinetics of Clonal yet Heterogeneous Enzymes." Journal of Physical Chemistry Letters 8, no. 13 (June 26, 2017): 3152–58. http://dx.doi.org/10.1021/acs.jpclett.7b01218.
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Carman, George M., Raymond A. Deems, and Edward A. Dennis. "Lipid Signaling Enzymes and Surface Dilution Kinetics." Journal of Biological Chemistry 270, no. 32 (August 11, 1995): 18711–14. http://dx.doi.org/10.1074/jbc.270.32.18711.
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Wu, H. B., and C. L. Tsou. "A comparison of Zn(II) and Co(II) in the kinetics of inactivation of aminoacylase by 1,10-phenanthroline and reconstitution of the apoenzyme." Biochemical Journal 296, no. 2 (December 1, 1993): 435–41. http://dx.doi.org/10.1042/bj2960435.
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The kinetics of reconstitution of apoacylase with either Zn(II) or Co(II) and the inactivation of the Co(II) reconstituted enzyme by 1,10-phenanthroline (OP) has been studied by following the substrate reaction continuously in presence of the metal ion or OP respectively. Although the native Zn(II)-containing and the Co(II)-reconstituted enzymes have closely similar Michaelis constants and maximal velocities, the kinetics for both the inactivation by OP and the reconstitution of the apoenzyme with the metal ions differs considerably. For Co(II), both the inactivation by OP and the reconstitution show simple kinetics, but for Zn(II), the inhibition by OP is a multi-phasic process [Wang, Wu, Wang, Zhou and Tsou (1992) Biochem. J. 281, 285-290], and the kinetics of reconstitution is also much more complicated. Both the native and the Co(II)-reconstituted enzymes are inhibited by excess of Zn(II), but not by Co(II). The inhibition by Zn(II) in excess and the reconstitution of the apoenzyme with Zn(II) are co-operative processes. The inhibition by Zn and its effect on the fluorescence emission of 1-anilinonaphthalene-8-sulphonic acid bound to the native enzyme indicate multiple Zn(II)-binding sites.
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Coggins, Si'Ana A., Bijan Mahboubi, Raymond F. Schinazi, and Baek Kim. "Mechanistic cross-talk between DNA/RNA polymerase enzyme kinetics and nucleotide substrate availability in cells: Implications for polymerase inhibitor discovery." Journal of Biological Chemistry 295, no. 39 (July 31, 2020): 13432–43. http://dx.doi.org/10.1074/jbc.rev120.013746.
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Enzyme kinetic analysis reveals a dynamic relationship between enzymes and their substrates. Overall enzyme activity can be controlled by both protein expression and various cellular regulatory systems. Interestingly, the availability and concentrations of intracellular substrates can constantly change, depending on conditions and cell types. Here, we review previously reported enzyme kinetic parameters of cellular and viral DNA and RNA polymerases with respect to cellular levels of their nucleotide substrates. This broad perspective exposes a remarkable co-evolution scenario of DNA polymerase enzyme kinetics with dNTP levels that can vastly change, depending on cell proliferation profiles. Similarly, RNA polymerases display much higher Km values than DNA polymerases, possibly due to millimolar range rNTP concentrations found in cells (compared with micromolar range dNTP levels). Polymerases are commonly targeted by nucleotide analog inhibitors for the treatments of various human diseases, such as cancers and viral pathogens. Because these inhibitors compete against natural cellular nucleotides, the efficacy of each inhibitor can be affected by varying cellular nucleotide levels in their target cells. Overall, both kinetic discrepancy between DNA and RNA polymerases and cellular concentration discrepancy between dNTPs and rNTPs present pharmacological and mechanistic considerations for therapeutic discovery.
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Mantle, T. J. "Enzymes: Nature's Nanomachines." Biochemical Society Transactions 29, no. 2 (May 1, 2001): 331–36. http://dx.doi.org/10.1042/bst0290331.
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The development of enzyme kinetics, protein crystallography and NMR studies allows enzymecatalysed reactions to be described in terms of mechanistic chemistry, albeit applied to relatively enormous molecules. These nanomachines, which so inspired Drexler's Engines of Creation, have been working in biological systems for over three billion years and represent a useful knowledge base for our further understanding of mechanistic biology. They also provide a tantalizing glimpse into what may be the basis for novel technologies with industrial applications for the twenty-first century.
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Nisar, Kinza, Roheena Abullah, Afshan Kaleem, Mehwish Iqtedar, and Tehreema Iftikar. "Optimization and production kinetics for cellulases by wild and mutant strain of Thermomyces dupontii in stirred tank reactor." Acta Scientiarum. Technology 44 (March 11, 2022): e57664. http://dx.doi.org/10.4025/actascitechnol.v44i1.57664.
Full textAbstract:
The main bottleneck in fermentation technology is scaling up procedure of industrial enzymes according to the biological characteristics of the organism. The current study describes the production kinetics of cellulases in stirred tank reactor by using mutant and wild strains of T. dupontii. The fermentation span of both the strains in bioreactor was examined. It is was found in mutant strain of T. dupontii fermentation time required for optimum production was reduced to 48h as compared to 72h in wild strain. The kinetic studies also exhibited greater value of µ (h-1) in case of mutated strain in comparison with wild strain. The effects of some other critical factors like agitation intensity dissolve oxygen, aeration, temperature, size of inoculum and pH was estimated on enzyme production kinetics. The results shows maximum activity of cellulases was attained at 220 rpm, 15% dissolve oxygen, aeration rate 1.5 vvm, 55°C, 8 % inoculum size and pH 5.5 for both strains respectively. The higher values of enzyme production kinetics i.e. Yp/x, Qp, Qx and qp in STR in case of mutant strain indicates its superiority over wild strain of T. dupontii. Thus mutant thermophilic T. dupontii might be a potential candidate for industrial applications.