A single-molecule force spectroscopy nanosensor for the identification of new antibiotics and antimalarials
| Autor: | Jordi Robles, Xavier Sisquella, Javier Alguacil, Fausto Sanz, Xavier Fernàndez-Busquets, Dario Anselmetti, Santiago Imperial, Karel de Pourcq |
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| Jazyk: | angličtina |
| Rok vydání: | 2010 |
| Předmět: |
Stereochemistry
pyruvate malaria Biosensing Techniques Sensitivity and Specificity Biochemistry drug discovery Antimalarials chemistry.chemical_compound glyceraldehyde-3-phosphate Transferases Nanosensor Escherichia coli Genetics Humans Nanotechnology Molecule 1-deoxy-D-xylulose 5-phosphate synthase Molecular Biology chemistry.chemical_classification Molecular Structure ATP synthase biology Drug discovery Spectrum Analysis Force spectroscopy Substrate (chemistry) Enzymes Immobilized Anti-Bacterial Agents Enzyme chemistry biology.protein Glyceraldehyde 3-phosphate 2-C-methyl-D-erythritol-4-phosphate pathway Biotechnology |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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