Tesis doctoral de Francesc Xavier Sisquella Duran
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 between escherichia coli 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 illustrate the substrate sequentiality in dxs activity, and allow for the calculation of enzymatic parameters with single-molecule resolution. The dxs inhibitor fluoropyruvate has been detected in our smfs competition experiments at a concentration of 10 ¿m, improving by two orders of magnitude the sensitivity of conventional enzymatic methods. The binding of dxs to pyruvate is a twostep process with dissociation constants of koff = ca. 6.10 ¿ 10-4 s1 and 128 s1, and reaction lengths of xíY = 63.0 í¥ and 0.4 í¥. These results open the field for the biodiscovery of new compounds that are present in chemical libraries or natural product extracts at concentrations below the detection limits of current screening approaches.
Datos académicos de la tesis doctoral «A force spectroscopy approach based on enzyme inhibition for the identification of new antibiotics and antimalarials«
- Título de la tesis: A force spectroscopy approach based on enzyme inhibition for the identification of new antibiotics and antimalarials
- Autor: Francesc Xavier Sisquella Duran
- Universidad: Barcelona
- Fecha de lectura de la tesis: 21/05/2010
Dirección y tribunal
- Director de la tesis
- Xavier Fernández Busquets
- Tribunal
- Presidente del tribunal: jordi Robles brau
- dario Anselmetti (vocal)
- (vocal)
- (vocal)