| Abstract: | Several classes of substrate analogs of dihydroxy-acid dehydratase have been tested as inhibitors of this enzyme in an attempt to characterize its binding site and find what modifications in substrate structure lead to an affinity higher than that of the natural substrates. The substrate analogs were tested on dihydroxy-acid dehydratase from both spinach and Escherichia coli. One modification of the substrate that led to as much as a 1000-fold increase in binding affinity was replacement of the 3-hydroxyl group with a thiol. It has been shown previously that the 3-hydroxyl group of the substrate becomes a ligand for one Fe of the Fe-S clusters of these enzymes on binding to their active sites. It seems likely then that the tighter binding of the thiol containing analogs is due to the thiol group becoming a ligand to an iron of the Fe-S clusters of these enzymes. A second modification in substrate that led to as much as 1000-fold increase in binding affinity was the addition of a large lipophilic group. This suggests there is a large hydrophobic pocket or hydrophobic surface near the active site of dihydroxy-acid dehydratase. A modification in substrate that led to as much as a 50-fold increase in binding was the replacement of the carboxyl group of the substrate with phosphonate; however, this increase was limited to substrate analogs without a polar functionality on the carbon β to the phosphonate group. Bromopyruvate was found to irreversibly inactivate dihydroxy-acid dehydratase. Each good inhibitor we found was active on spinach dihydroxy-acid dehydratase and E. coli dihydroxy-acid dehydratase to a similar extent suggesting the active sites of the enzymes from these two organisms are similar. Some of the better inhibitors described in this report have mild herbicidal activity. |
