Using Substrate Analogues To Probe the Kinetic Mechanism and Active Site of Escherichia coli MenD
Citations Over TimeTop 20% of 2011 papers
Abstract
MenD catalyzes the thiamin diphosphate-dependent decarboxylative carboligation of α-ketoglutarate and isochorismate. The enzyme is essential for menaquinone biosynthesis in many bacteria and has been proposed to be an antibiotic target. The kinetic mechanism of this enzyme has not previously been demonstrated because of the limitations of the UV-based kinetic assay. We have reported the synthesis of an isochorismate analogue that acts as a substrate for MenD. The apparent weaker binding of this analogue is advantageous in that it allows accurate kinetic experiments at substrate concentrations near K(m). Using this substrate in concert with the dead-end inhibitor methyl succinylphosphonate, an analogue of α-ketoglutarate, we show that MenD follows a ping-pong kinetic mechanism. Using both the natural and synthetic substrates, we have measured the effects of 12 mutations of residues at the active site. The results give experimental support to previous models and hypotheses and allow observations unavailable using only the natural substrate.
Related Papers
- → Recombinant Protein Expression in Escherichia coli (E.coli): What We Need to Know(2018)115 cited
- [A study on beta-lactamase activity of biofilm Escherichia coli].(2001)
- Bacteriostatic Experiment of Twelve Species of Chinese Medicines and Its Compounds Against Escherichia Coli(2004)
- Damaging effect of pressurized CO_2 on cell membrane of Escherichia coli(2013)
- A study on β-lactamase activity of biofilm Escherichia coli(2001)