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A Fragment‐Based Approach to Understanding Inhibition of 1‐Deoxy‐ D ‐Xylulose‐5‐Phosphate Reductoisomerase
Author(s) -
Mercklé Ludovic,
de AndrésGómez Ana,
Dick Bethany,
Cox Russell J.,
Godfrey Christopher R. A.
Publication year - 2005
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200500061
Subject(s) - cooperativity , cooperative binding , chemistry , phosphate buffered saline , stereochemistry , binding site , substrate (aquarium) , enzyme , biochemistry , biology , chromatography , ecology
The inhibition of 1‐deoxy‐ D ‐xylulose‐5‐phosphate reductoisomerase (DXR) by fosmidomycin was studied by using a kinetic assay based on the consumption of NADPH and synthetic substrate. Fosmidomycin is a slow tight‐binding inhibitor of DXR that shows strong negative cooperativity (| h |=0.3) in binding. Cooperativity is displayed during the initial (weak, K 0.5 =10 μ M ) binding event and does not change as the binding tightens to the equilibrium value of 0.9 n M over a period of seconds to minutes. A series of fosmidomycin fragments was examined, but all showed much weaker inhibition, in the m M range. A series of cyclic fosmidomycin analogues was also synthesised and tested, but these showed high‐μ M binding at best. None of the synthetic compounds showed time‐dependent inhibition. We concluded that the slow tight‐binding behaviour, and perhaps also cooperativity, are mediated by significant reorganisation of the active site upon fosmidomycin binding. This makes the rational design of new inhibitors of DXR difficult at best.