Future research will expand on two questions raised by previous work. The process by which enzymes liberate their products into the medium, rarely accessible to investigation, has become the central issue of our ongoing kinetic studies of the fumarase reaction. The active site (A-site) of the enzyme sits at the bottom of a well from which the product, fumarate, F, must escape to complete the reaction. Recent structural studies identified a second molecule of substrate, situated at the top of the well, the B-site. The role of the B-site is unknown but has been used to explain the activation observed with high concentrations of substrate, malate. Our studies with a B-site mutant suggest that product release to the medium occurs via the B-site. The A-site is then available to substrate again and helps the product leave the B-site.II. Previous work has suggested that the use of glutathione in the glyoxalase enzyme system may have evolved by virtue of its role in activating a prior chemical step. This suggested to us that cells that do not contain glutathione such as mycobacteria could employ their major thiol, mycothiol, instead of GSH for the nonenzymatic step and lead to an MSH based glyoxalase system. We will establish the chemistry of the glyoxalase system of Mycobacter smegmatis. Since methylglyoxal is toxic to cells and must be removed by an active glyoxalse system, inhibition of a MSH based system could result in a treatment for many mycobacterial-based diseases such as tuberculosis.
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