Escherichia coli phosphoenolpyruvate carboxylase: Effect of allosteric inhibitors on the kinetic parameters and sedimentation behavior

Abstract

Various metabolic intermediates and chemically related compounds were studied to determine their effects on the catalytic activity and sedimentation behavior in sucrose gradients of phosphoenolpyruvate carboxylase of Escherichia coli. Two types of enzyme-inhibitor interactions were found. In the first type of inhibition, the inhibitor was functionally competitive with acetyl coenzyme A and had the ability to stabilize the tetrameric form of the enzyme, as evidenced by its effect on the apparent sedimentation rate of the enzyme in sucrose gradients. These compounds were approximately equivalent in length to four-carbon dicarboxylic acids and had to be ionized with negative charges at both ends. In addition, the strong inhibitors (aspartate, malate, fumarate, cysteine, and tartrate) have an electron-attracting group as an L-α substituent. Fumarate was also a strong inhibitor, and it too contained an electron-attracting group in the form of an olefinic bond. The second type of inhibition was exhibited uniquely by maleate. Maleate inhibited the enzyme strongly, but it was not competitive with acetyl coenzyme A nor could it stabilize the tetrameric form of the enzyme at concentrations that were highly inhibitory. The pattern of inhibition by maleate could be converted to that seen with the other inhibitors in the presence of aspartate. This indicates that each class of inhibitor is competitive with the other and that all allosteric inhibitors may share the same binding site.