Kinetic and thermodynamic characterization of the RNA guanylyltransferase reaction

An RNA guanylyltransferase activity is involved in the synthesis of the cap structure found at the 5' end of eukaryotic mRNAs. The RNA guanylyltransferase activity is a two-step ping-pong reaction in which the enzyme first reacts with GTP to produce the enzyme-GMP covalent intermediate with the concomitant release of pyrophosphate. In the second step of the reaction, the GMP moiety is then transferred to a diphosphorylated RNA. Both reactions were previously shown to be reversible. In this study, we report a biochemical and thermodynamic characterization of both steps of the reaction of the RNA guanylyltransferase from Paramecium bursaria Chlorella virus 1, the prototype of a family of viruses infecting green algae. Using a combination of real-time fluorescence spectroscopy, radioactive kinetic assays, and inhibition assays, the complete kinetic parameters of the RNA guanylyltransferase were determined. We produced a thermodynamic scheme for the progress of the reaction as a function of the energies involved in each step. We were able to demonstrate that the second step comprises the limiting steps for both the direct and reverse overall reactions. In both cases, the binding to the RNA substrates is the step requiring the highest energy and generating unstable intermediates that will promote the catalytic activites of the enzyme. This study reports the first thorough kinetic and thermodynamic characterization of the reaction catalyzed by an RNA capping enzyme.     

A molecular mechanism for the production of muscular work

Motility in biological systems is widely thought to result from the transduction of chemical free energy. In muscle a difficulty has been encountered in finding a precise mechanism whereby this conversion is accomplished. We suggest that this difficulty resides in the macroscopic character of free energy which, as a thermodynamic quantity, deals only with large assemblages of molecules. However, the fundamental site of active movement has recently been found to be localized in a single molecule (a myosin head) and is therefore not open to thermodynamic treatment. It is suggested instead that the energetic source of work produced at the myosin head is to be found in the heat (in the form of kinetic energy) evolved during an actomyosin ATPase cycle. This heat equivalent kinetic energy is then converted into useful work by means of a vibrational mode of a single water molecule which is attached to the ADP formed in the myosin head during a portion of the actomyosin ATPase cycle. It is the resonance mode of this water molecule which enables the extremely short durations (10^-15s) of the chemical reactions taking place in one actomyosin ATPase cycle to result in the much longer duration (10^-2s) of the resulting movement. This mechanism may also be fundamental to other types of motility in living systems.     

Thermodynamic activity‐based intrinsic enzyme kinetic sheds light on enzyme–solvent interactions

The reaction medium has major impact on biocatalytic reaction systems and on their economic significance. To allow for tailored medium engineering, thermodynamic phenomena, intrinsic enzyme kinetics, and enzyme–solvent interactions have to be discriminated. To this end, enzyme reaction kinetic modeling was coupled with thermodynamic calculations based on investigations of the alcohol dehydrogenase from Lactobacillus brevis (LbADH) in monophasic water/methyl tert‐butyl ether (MTBE) mixtures as a model solvent. Substrate concentrations and substrate thermodynamic activities were varied separately to identify the individual thermodynamic and kinetic effects on the enzyme activity. Microkinetic parameters based on concentration and thermodynamic activity were derived to successfully identify a positive effect of MTBE on the availability of the substrate to the enzyme, but a negative effect on the enzyme performance. In conclusion, thermodynamic activity‐based kinetic modeling might be a suitable tool to initially curtail the type of enzyme–solvent interactions and thus, a powerful first step to potentially understand the phenomena that occur in nonconventional media in more detail. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:96–103, 2017     

The Glansdorff-Prigogine thermodynamic stability criterion in the light of Lyapunov's theory.

The significance of thermodynamic coupling in chemical reactions—which recently has been questioned on thermodynamic grounds—is examined from the point of view of kinetics. It is shown that considerations of stoichiometry lead to a meaningful definition of velocity for elementary and certain elementary-complex reactions, whereas the non-equilibrium thermodynamic definition of reaction velocity is ambiguous in multireaction systems. Based on rate laws which include the effects of nonideality, it is proven that elementary-type reactions are not coupled thermodynamically and concluded that thermodynamic coupling has no kinetic significance. A discussion is given to show that this result is compatible with coupling in both biochemical systems and oscillating reactions.     

The driving force for life's emergence: kinetic and thermodynamic considerations

The principles that govern the emergence of life from non-life remain a subject of intense debate. The evolutionary paradigm built up over the last 50 years, that argues that the evolutionary driving force is the Second Law of Thermodynamics, continues to be promoted by some, while severely criticized by others. If the thermodynamic drive toward ever-increasing entropy is not what drives the evolutionary process, then what does? In this paper, we analyse this long-standing question by building on Eigen's "replication first" model for life's emergence, and propose an alternative theoretical framework for understanding life's evolutionary driving force. Its essence is that life is a kinetic phenomenon that derives from the kinetic consequences of autocatalysis operating on specific biopolymeric systems, and this is demonstrably true at all stages of life's evolution--from primal to advanced life forms. Life's unique characteristics--its complexity, energy-gathering metabolic systems, teleonomic character, as well as its abundance and diversity, derive directly from the proposition that from a chemical perspective the replication reaction is an extreme expression of kinetic control, one in which thermodynamic requirements have evolved to play a supporting, rather than a directing, role. The analysis leads us to propose a new sub-division within chemistry--replicative chemistry. A striking consequence of this kinetic approach is that Darwin's principle of natural selection: that living things replicate, and therefore evolve, may be phrased more generally: that certain replicating things can evolve, and may therefore become living. This more general formulation appears to provide a simple conceptual link between animate and inanimate matter.     

Thermodynamic Constraints in Kinetic Modeling: Thermodynamic‐Kinetic Modeling in Comparison to Other Approaches

Kinetic models of reaction networks may easily violate the laws of thermodynamics and the principle of detailed balance. In large network models, the constraints that are imposed by these laws are particularly difficult to address. This hinders modeling of biochemical reaction networks. Thermodynamic‐kinetic modeling is a method that provides a thermodynamically sound and formally appealing way for deriving dynamic model equations of reaction systems. State variables of this approach are thermokinetic potentials that describe the ability of compounds to drive a reaction. A compound has a parameter called capacity, which is the ratio of its concentration and thermokinetic potential. A reaction is described by its resistance which is the ratio of the thermokinetic driving force and flux. In these aspects, the formalism is similar to the modeling formalism for electrical networks and an analogous graphical representation is possible. The thermodynamic‐kinetic modeling formalism is equivalent to the traditional kinetic modeling formalism with the exception that it is not possible to build thermodynamically infeasible models. Here, the thermodynamic‐kinetic modeling formalism is reviewed, compared to other approaches, and some of its advantages are worked out. In contrast to other approaches, thermodynamic‐kinetic modeling does not rely on an explicit enumeration of stoichiometric cycles. It is capable of describing rate laws far from equilibrium. Further, the parameterization by capacities and resistances is particularly intuitive and powerful.     

Dissipative Structures for an Allosteric Model: Application to Glycolytic Oscillations

An allosteric model of an open monosubstrate enzyme reaction is analyzed for the case where the enzyme, containing two protomers, is activated by the product. It is shown that this system can lead to instabilities beyond which a new state organized in time or in space (dissipative structure) can be reached. The conditions for both types of instabilities are presented and the occurrence of a temporal structure, consisting of a limit cycle behavior, is determined numerically as a function of the important parameters involved in the system. Sustained oscillations in the product and substrate concentrations are shown to occur for acceptable values of the allosteric and kinetic constants; moreover, they seem to be favored by substrate activation. The model is applied to phosphofructokinase, which is the enzyme chiefly responsible for glycolytic oscillations and which presents the same pattern of regulation as the allosteric enzyme appearing in the model. A qualitative and quantitative agreement is obtained with the experimental observations concerning glycolytic self-oscillations.     

Extraction of astaxanthin from giant tiger (Panaeus monodon) shrimp waste using palm oil: studies of extraction kinetics and thermodynamic

Study of extraction of astaxanthin from giant tiger (Panaeus monodon) shrimp waste using palm oil was conducted to determine the extraction kinetics and thermodynamic parameters. Two extraction models were proposed: mass transfer kinetic model and reaction kinetic model. It was found that both of mass transfer and reaction kinetic control the extraction of astaxanthin from shrimp waste using palm oil. The thermodynamic parameters of extraction were also obtained in this study.     

Kinetic manifestations of slow conformation rearrangements of lactate dehydrogenase. An experiment and a mathematical model

Non-steady-state kinetics of lactate dehydrogenase (LDH) catalyzed reaction was investigated for a wide time interval (from 100 msec to 1-3 min) by using stopped-flow methods. A two-stage character of LDH reaction, slow changes like a lag-period on kinetic curves at pH 8.0, flexions on kinetic curves after pre-mixing LDH with NAD+ and pyruvate have been revealed. The graph theory for mathematical analysis of experimental data was applied, which has been developed for the non-steady-state kinetics. An enzyme model of the two-conformer LDH structure was used. The reaction scheme with a preferential inhibition of one of the conformers (pH 8.0) is suggested. The obtained values of kinetic constants prove that transitions between LDH conformers must be slow.     

The ATPase cycle mechanism of the DEAD-box rRNA helicase, DbpA

DEAD-box proteins are ATPase enzymes that destabilize and unwind duplex RNA. Quantitative knowledge of the ATPase cycle parameters is critical for developing models of helicase activity. However, limited information regarding the rate and equilibrium constants defining the ATPase cycle of RNA helicases is available, including the distribution of populated biochemical intermediates, the catalytic step(s) that limits the enzymatic reaction cycle, and how ATP utilization and RNA interactions are linked. We present a quantitative kinetic and equilibrium characterization of the ribosomal RNA (rRNA)-activated ATPase cycle mechanism of DbpA, a DEAD-box rRNA helicase implicated in ribosome biogenesis. rRNA activates the ATPase activity of DbpA by promoting a conformational change after ATP binding that is associated with hydrolysis. Chemical cleavage of bound ATP is reversible and occurs via a γ-phosphate attack mechanism. ADP-P_i and RNA binding display strong thermodynamic coupling, which causes DbpA-ADP-P_i to bind rRNA with > 10-fold higher affinity than with bound ATP, ADP or in the absence of nucleotide. The rRNA-activated steady-state ATPase cycle of DbpA is limited both by ATP hydrolysis and by P_i release, which occur with comparable rates. Consequently, the predominantly populated biochemical states during steady-state cycling are the ATP- and ADP-P_i-bound intermediates. Thermodynamic linkage analysis of the ATPase cycle transitions favors a model in which rRNA duplex destabilization is linked to strong rRNA and nucleotide binding. The presented analysis of the DbpA ATPase cycle reaction mechanism provides a rigorous kinetic and thermodynamic foundation for developing testable hypotheses regarding the functions and molecular mechanisms of DEAD-box helicases.     

Laser excitation studies of the product release steps in the catalytic cycle of the light-driven enzyme, protochlorophyllide oxidoreductase

The latter stages of the catalytic cycle of the light-driven enzyme, protochlorophyllide oxidoreductase, have been investigated using novel laser photoexcitation methods. The formation of the ternary product complex was initiated with a 6-ns laser pulse, which allowed the product release steps to be kinetically accessed for the first time. Subsequent absorbance changes associated with the release of the NADP+ and chlorophyllide products from the enzyme could be followed on a millisecond timescale. This has facilitated a detailed kinetic and thermodynamic characterization for the interconversion of all the various bound and unbound product species. Initially, NADP+ is released from the enzyme in a biphasic process with rate constants of 1210 and 237 s(-1). The rates of both phases show a significant dependence on the viscosity of the solvent and become considerably slower at higher glycerol concentrations. The fast phase of this process exhibits no dependence on NADP+ concentration, suggesting that conformational changes are required prior to NADP+ release. Following NADP+ release, the NADPH rebinds to the enzyme with a maximum rate constant of approximately 72 s(-1). At elevated temperatures (>298 K) chlorophyllide is released from the enzyme to yield the free product with a maximum rate constant of 20 s(-1). The temperature dependencies of the rates of each of these steps were measured, and enthalpies and entropies of activation were calculated using the Eyring equation. A comprehensive kinetic and thermodynamic scheme for these final stages of the reaction mechanism is presented.     

Theoretical discussion on the mechanism of binding CO2 by DBU and alcohol

Quantum chemical studies on the reaction of binding CO₂ by amidine base diazabicyclo [5.4.0]-undec-7-ene (DBU) and alcohol were carried out at the B3LYP/6-31g(d) level in order to find the reaction mechanism. The structures of reactants and product were optimised, and thermodynamic analyses were also carried out using the single point energy calculation and frequency analyses. It is noted that the reaction of binding CO₂ by DBU and propanol is thermodynamically feasible and qualitatively in accordance with the experimental observations. The results of thermodynamic and kinetic analyses demonstrate that the possible reaction mechanisms can be a two-step bimolecular reaction and a one-step trimolecular reaction. In the two-step bimolecular mechanism, the first step is the formation of intermediate by DBU and CO₂, and the second step is the nucleophilic attack of propanol on the intermediate. In the one-step trimolecular mechanism, O and H atoms of hydroxyl in propanol form an O–C bond with CO₂ and an H–N bond with DBU, respectively. The one-step trimolecular reaction seems a more reasonable mechanism because of the consideration of kinetic parameters.     

Chromatophore heterogeneity explains phenomena seen in Rhodobacter sphaeroides previously attributed to supercomplexes

It is generally considered that metabolic reactions are well described by homogeneous kinetic models in which the reaction phase is statistically uniform. In membranes, especially in photosynthetic systems where the protein complement is high, it has recently been recognized that effects of local heterogeneity might contribute additional factors that perturb the kinetic behavior, and require more extensive treatment. We show in this paper that statistical heterogeneity in vesicular systems can also contribute to quite marked discrepancies from the behavior expected from standard kinetic and thermodynamic models, for reactions involving free diffusion in the aqueous phase. We explain the kinetic and thermodynamic effects observed in studies of photosynthetic electron transfer in cells and chromatophores from Rhodobacter sphaeroides previously attributed to supercomplexes, in terms of a model based on heterogeneity in distribution of electron transfer components among the chromatophore population. We discuss examples of data inconsistent with the supercomplex model, but well explained by the heterogeneity model.     

Simplified model for Belousov-Zhabotinsky reaction

Noyes' scheme of the elementary processes for the Belousov-Zhabotinsky reaction has been simplified on the basis of reaction kinetic consideration. The simplest possible analogue (model K) has been described by a set of kinetic equations, and it is solved to examine the varieties of new ordered phases, which include temporal rhythm and spatial pattern. Particular attention has been given to the onset of new phases, which is associated with an anomalous enhancement of fluctuations. A stochastic theory of fluctuations, which was developed in our previous work, has been applied to the present case. Theoretical results compare reasonably well with experimental findings, i.e. with (1) spatial periodic structure and (2) limit cycle behaviour.     

Natural selection for kinetic stability is a likely origin of correlations between mutational effects on protein energetics and frequencies of amino acid occurrences in sequence alignments

It appears plausible that natural selection constrains, to some extent at least, the stability in many natural proteins. If, during protein evolution, stability fluctuates within a comparatively narrow range, then mutations are expected to be fixed with frequencies that reflect mutational effects on stability. Indeed, we recently reported a robust correlation between the effect of 27 conservative mutations on the thermodynamic stability (unfolding free energy) of Escherichia coli thioredoxin and the frequencies of residues occurrences in sequence alignments. We show here that this correlation likely implies a lower limit to thermodynamic stability of only a few kJ/mol below the unfolding free energy of the wild-type (WT) protein. We suggest, therefore, that the correlation does not reflect natural selection of thermodynamic stability by itself, but of some other factor which is linked to thermodynamic stability for the mutations under study. We propose that this other factor is the kinetic stability of thioredoxin in vivo, since( i) kinetic stability relates to irreversible denaturation, (ii) the rate of irreversible denaturation in a crowded cellular environment (or in a harsh extracellular environment) is probably determined by the rate of unfolding, and (iii) the half-life for unfolding changes in an exponential manner with activation free energy and, consequently, comparatively small free energy effects can have deleterious consequences for kinetic stability. This proposal is supported by the results of a kinetic study of the WT form and the 27 single-mutant variants of E. coli thioredoxin based on the global analyses of chevron plots and equilibrium unfolding profiles determined from double-jump unfolding assays. This kinetic study suggests, furthermore, one of the factors that may contribute to the high activation free energy for unfolding in thioredoxin (required for kinetic stability), namely the energetic optimization of native-state residue environments in regions, which become disrupted in the transition state for unfolding.     

Instability and oscillatory behavior of membrane-chemical reaction systems

The stability problem of a chemical system consisting of reversible reactions is analyzed with the aid of computer calculations. The system is based on the model proposed by Edelstein and exhibits oscillations of chemical species. The analysis shows that the oscillatory character is of limit cycle type. The results are applied to the construction of a membrane-chemical reaction system, which shows characteristic instability behavior. This is useful as a model of cell division.     

The physical properties of an effective lung surfactant

It is suggested that the phospholipids at the alveolar/air interface exhibit both thermodynamic (equilibrium) and kinetic forces during the course of a respiratory cycle. The alveolae are kept open at full expiration by a residue of nearly pure dipalmitoyl phosphatidylcholine which is condensed and therefore, incompressible at 37 degrees C.     

Esterification by immobilized lipase in solvent-free media: kinetic and thermodynamic arguments

The aim of this study is to characterize, in solvent-free systems (SFS), the kinetic and thermodynamic performance of batch lipase-catalyzed esterification. SFS are compared to a conventional organic solvent, n-hexane. The esterification of oleic acid with ethanol was chosen as a model reaction. The TABEK (thermodynamic activity-based enzyme kinetics) approach was used to rationally analyze kinetics. Influence of the reaction medium on final conversions was also studied. Several factors, such as initial molar ratio of substrates, reactant availability, initial water content, and quantity of immobilized enzyme, were examined. Special attention was also turned to enzyme stability and reuse after reaction, this last item being a prerequisite in the development of industrial processes. SFS proved to be almost as efficient as n-hexane from a kinetic and thermodynamic point of view and offered a better volumetric production.     

Reducing intrinsic biochemical noise in cells and its thermodynamic limit

In living cells, the specificity of biomolecular recognition can be amplified and the noise from non-specific interactions can be reduced at the expense of cellular free energy. This is the seminal idea in the Hopfield-Ninio theory of kinetic proofreading: The specificity is increased via cyclic network kinetics without altering molecular structures and equilibrium affinites. We show a thermodynamic limit of the specificity amplification with a given amount of available free energy. For a normal cell under physiological condition with sustained phosphorylation potential, this gives a factor of 10(10) as the upper bound in specificity amplification. We also study an optimal kinetic network design that is capable of approaching the thermodynamic limit.