A potential role for isothermal calorimetry in studies of the effects of thermodynamic non‐ideality in enzyme‐catalyzed reactions

Lonhienne, Thierry; Winzor, Donald J.

doi:10.1002/jmr.706

Cited by 14 publications

(13 citation statements)

References 68 publications

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“…The ITC method that has been extensively reviewed [19][20][21][22][23][24][25][26] will not be covered. DSC is much used for the study of the stability of biological macromolecules and the folding of proteins, but it can also be applied in order to understand biomolecular interactions and can thus be an interesting technique in the process of drug design [27].…”

Section: Differential Scanning Calorimetrymentioning

confidence: 99%

Differential Scanning Calorimetry in Life Science: Thermodynamics, Stability, Molecular Recognition and Application in Drug Design

Bruylants¹,

Wouters²,

Michaux

2005

CMC

297

191

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All biological phenomena depend on molecular recognition, which is either intermolecular like in ligand binding to a macromolecule or intramolecular like in protein folding. As a result, understanding the relationship between the structure of proteins and the energetics of their stability and binding with others (bio)molecules is a very interesting point in biochemistry and biotechnology. It is essential to the engineering of stable proteins and to the structure-based design of pharmaceutical ligands. The parameter generally used to characterize the stability of a system (the folded and unfolded state of the protein for example) is the equilibrium constant (K) or the free energy (deltaG(o)), which is the sum of enthalpic (deltaH(o)) and entropic (deltaS(o)) terms. These parameters are temperature dependent through the heat capacity change (deltaCp). The thermodynamic parameters deltaH(o) and deltaCp can be derived from spectroscopic experiments, using the van't Hoff method, or measured directly using calorimetry. Along with isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) is a powerful method, less described than ITC, for measuring directly the thermodynamic parameters which characterize biomolecules. In this article, we summarize the principal thermodynamics parameters, describe the DSC approach and review some systems to which it has been applied. DSC is much used for the study of the stability and the folding of biomolecules, but it can also be applied in order to understand biomolecular interactions and can thus be an interesting technique in the process of drug design.

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Section: Differential Scanning Calorimetrymentioning

confidence: 99%

Differential Scanning Calorimetry in Life Science: Thermodynamics, Stability, Molecular Recognition and Application in Drug Design

Bruylants¹,

Wouters²,

Michaux

2005

CMC

297

191

View full text Add to dashboard Cite

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“…Evaluation of the Calorimetric Assay Calorimetry has been previously proven successful to characterize the activity of enzymes in dilute aqueous solutions [31][32][33], in complex crowded systems [34,35], and water-rich suspensions of insoluble subtrates [36,37] including hydrolysis of lignocellulose [19]. In all of these studies, calorimetry has been applied in the "titration mode", where enzyme and substrate are mixed by injections under continuous stirring.…”

Section: Resultsmentioning

confidence: 99%

Kinetics of Enzymatic High-Solid Hydrolysis of Lignocellulosic Biomass Studied by Calorimetry

Olsen

Lumby

McFarland

et al. 2010

Appl Biochem Biotechnol

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Enzymatic hydrolysis of high-solid biomass (>10% w/w dry mass) has become increasingly important as a key step in the production of second-generation bioethanol. To this end, development of quantitative real-time assays is desirable both for empirical optimization and for detailed kinetic analysis. In the current work, we have investigated the application of isothermal calorimetry to study the kinetics of enzymatic hydrolysis of two substrates (pretreated corn stover and Avicel) at high-solid contents (up to 29% w/w). It was found that the calorimetric heat flow provided a true measure of the hydrolysis rate with a detection limit of about 500 pmol glucose s(-1). Hence, calorimetry is shown to be a highly sensitive real-time method, applicable for high solids, and independent on the complexity of the substrate. Dose-response experiments with a typical cellulase cocktail enabled a multidimensional analysis of the interrelationships of enzyme load and the rate, time, and extent of the reaction. The results suggest that the hydrolysis rate of pretreated corn stover is limited initially by available attack points on the substrate surface (<10% conversion) but becomes proportional to enzyme dosage (excess of attack points) at later stages (>10% conversion). This kinetic profile is interpreted as an increase in polymer end concentration (substrate for CBH) as the hydrolysis progresses, probably due to EG activity in the enzyme cocktail. Finally, irreversible enzyme inactivation did not appear to be the source of reduced hydrolysis rate over time.

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“…A milestone in macromolecular crowding research was the development of the method called non-ideal tracer sedimentation equilibrium [35], which made feasible the analysis of the oligomerization of a labeled dilute species (tracer), in presence of non-related macromolecules acting as crowders. In the last decade, methods such as nuclear magnetic resonance and circular dichroism spectroscopies, as well as isothermal titration calorimetry have also been used to explore protein conformation, reaction kinetics and protein diffusion in crowding conditions [19,22,[36][37][38][39][40][41][42]. Finally, some examples can be found of fluorescence spectroscopy based determinations in macromolecular crowded solutions [43][44][45][46][47][48].…”

Section: Introductionmentioning

confidence: 99%

Quantitative Investigation of Biomolecular Interactions in Crowded Media by Fluorescence Spectroscopy, a Good Choice

Zorrilla

Lillo²

2009

CPPS

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Fluorescence spectroscopy methods have been proved to be powerful tools for the quantitative investigation of homologous and heterologous interactions, rotational and translational diffusion, and structural dynamics of biological molecules in crowded media. In addition to their high sensitivity, these methods present the advantage that the selective fluorescent labeling of the biomolecules under study allows distinguishing them from the background species. Moreover, the recent development of biological applications of single molecule fluorescence micro-spectroscopy methods has opened the possibility of performing quantitative determinations inside cells. In the last decades, theoretical and experimental studies have demonstrated the possible influence of the high concentration of macromolecules within living systems, on the thermodynamics and kinetics of biological reactions. Therefore, there is a growing interest in quantitatively evaluating the interactions involving biomolecules in the natural environment in which they occur. Since this is not always feasible, experiments conducted in model crowded conditions, resembling physiological media, may contribute to reduce the gap between traditional in vitro and in vivo experiments. In this review we will discuss the application of some fluorescence spectroscopy approaches, for the identification and quantification of biological macromolecules and their functional interactions in model crowded conditions.

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A potential role for isothermal calorimetry in studies of the effects of thermodynamic non‐ideality in enzyme‐catalyzed reactions

Cited by 14 publications

References 68 publications

Differential Scanning Calorimetry in Life Science: Thermodynamics, Stability, Molecular Recognition and Application in Drug Design

Differential Scanning Calorimetry in Life Science: Thermodynamics, Stability, Molecular Recognition and Application in Drug Design

Kinetics of Enzymatic High-Solid Hydrolysis of Lignocellulosic Biomass Studied by Calorimetry

Quantitative Investigation of Biomolecular Interactions in Crowded Media by Fluorescence Spectroscopy, a Good Choice

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