Estimating Kinetic and Thermodynamic Parameters from Single Molecule Enzyme−Inhibitor Interactions

Porter-Peden, Laura; Kamper, Sarah; Wal, Mark Vander; Blankespoor, Ronald L.; Sinniah, Kumar

doi:10.1021/la801477a

Cited by 19 publications

(13 citation statements)

References 22 publications

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“…The planned configuration of SMFS assays consisted of DXS‐functionalized AFM cantilevers and pyruvate‐or G3P‐functionalized surfaces via heterobifunctional linkers (32). Such strategy introduces a distance between interacting molecules and surfaces, adds steric flexibility for the binding partners, and guarantees an almost complete reduction of unspecific binding events (53), although it might affect the calculation of the apparent kinetic and thermodynamic enzymatic parameters (23). Because E. coli DXS has 36 lysines, of which only K289 is present in the active center (54), we followed established protocols (48) for the covalent linkage of the enzyme to SiN 3 cantilevers through the lateral amino group in lysine residues, using a monodisperse ∼100 nm‐long polyethylene glycol (PEG) linker ( Fig.…”

Section: Resultsmentioning

confidence: 99%

A single‐molecule force spectroscopy nanosensor for the identification of new antibiotics and antimalarials

et al. 2010

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An important goal of nanotechnology is the application of individual molecule handling techniques to the discovery of potential new therapeutic agents. Of particular interest is the search for new inhibitors of metabolic routes exclusive of human pathogens, such as the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway essential for the viability of most human pathogenic bacteria and of the malaria parasite. Using atomic force microscopy single-molecule force spectroscopy (SMFS), we have probed at the single-molecule level the interaction of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), which catalyzes the first step of the MEP pathway, with its two substrates, pyruvate and glyceraldehyde-3-phosphate. The data obtained in this pioneering SMFS analysis of a bisubstrate enzymatic reaction illustrate the substrate sequentiality in DXS activity and allow for the calculation of catalytic parameters with single-molecule resolution. The DXS inhibitor fluoropyruvate has been detected in our SMFS competition experiments at a concentration of 10 μM, improving by 2 orders of magnitude the sensitivity of conventional enzyme activity assays. The binding of DXS to pyruvate is a 2-step process with dissociation constants of k(off) = 6.1 × 10(-4) ± 7.5 × 10(-3) and 1.3 × 10(-2) ± 1.0 × 10(-2) s(-1), and reaction lengths of x(β) = 3.98 ± 0.33 and 0.52 ± 0.23 Å. These results constitute the first quantitative report on the use of nanotechnology for the biodiscovery of new antimalarial enzyme inhibitors and open the field for the identification of compounds represented only by a few dozens of molecules in the sensor chamber.

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Section: Resultsmentioning

confidence: 99%

A single‐molecule force spectroscopy nanosensor for the identification of new antibiotics and antimalarials

et al. 2010

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“…Beginning with the pioneering work on complementary DNA strands [8], biotin-streptavidin [9,10] and cell adhesion proteoglycans [11], this method has been applied to a wide range of interactions: from complex biological ones such as antibody-antigen [12][13][14], protein-nucleic acid [15][16][17][18][19], quadruplex nucleic acids [20], enzymeinhibitor [21] and cell adhesion molecules [22][23][24], to synthetic biology [25] and supramolecular examples [26]. The results of these experiments demonstrate that the technique can be applied to ligand-receptor interactions with dissociation constants, K D , ranging from fM to µM with point mutant sensitivity.…”

Section: Technology Reportmentioning

confidence: 99%

Single-Molecule Force Spectroscopy: A Method for Quantitative Analysis of Ligand–Receptor Interactions

Fuhrmann

Ros²

2010

Nanomedicine

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The quantitative analysis of molecular interactions is of high interest in medical research. Most methods for the investigation of ligand-receptor complexes deal with huge ensembles of biomolecules, but often neglect interactions with low affinity or small subpopulations with different binding properties. Single-molecule force spectroscopy offers fascinating possibilities for the quantitative analysis of ligand-receptor interactions in a wide affinity range and the sensitivity to detect point mutations. Furthermore, this technique allows one to address questions about the related binding energy landscape. In this article, we introduce single-molecule force spectroscopy with a focus on novel developments in both data analysis and theoretical models for the technique. We also demonstrate two examples of the capabilities of this method.

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“…Fitting the force spectrum data to different theoretical models will yield kinetic and thermodynamic information for the single‐molecule interaction. The Bell‐Evans equation is the most commonly used model (Porter‐Peden and others 2008).…”

Section: Discussionmentioning

confidence: 99%

Mechanochemistry in Thermomechanical Processing of Foods: Kinetic Aspects

Zhao¹,

Wei²,

Wang³

et al. 2011

Journal of Food Science

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In some food processing operations, raw materials sustain shearing and heating simultaneously. These thermomechanical treatments will induce chemical, physicochemical, and biochemical changes, such as starch degradation and gelatinization, protein denaturalization, loss of nutritious components, and inactivation of enzymes and their inhibitors. In this article, only kinetic aspects of these changes are reviewed. The Basedow and Zhurkov models are commonly used to describe shear effects on the rate constants of mechanochemical reactions. After a brief description of these 2 models, their applications in food are reviewed deeply, with emphasis on the activation energy reduction in the Zhurkov model and the shear activation energy in the Basedow model. Since the changes occurring in food systems often involve various interactions at the atomic, molecular, and supramolecular levels, they were described only phenomenonlogically by the mechanochemical kinetic models. The limitation, opportunity, and extension of the mechanochemical approach to modeling food kinetics are discussed.

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Estimating Kinetic and Thermodynamic Parameters from Single Molecule Enzyme−Inhibitor Interactions

Cited by 19 publications

References 22 publications

A single‐molecule force spectroscopy nanosensor for the identification of new antibiotics and antimalarials

A single‐molecule force spectroscopy nanosensor for the identification of new antibiotics and antimalarials

Single-Molecule Force Spectroscopy: A Method for Quantitative Analysis of Ligand–Receptor Interactions

Mechanochemistry in Thermomechanical Processing of Foods: Kinetic Aspects

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