Allosteric enzymes are regulated by effectors or ligands that bind at a site other than their active site. Allosteric proteins are generally composed of 2 or more subunits and exist in two states: an active, high affinity (R state, stabilized by activators) and an inactive, low affinity substrate binding states (T state, stabilized by inhibitors). When binding of a ligand (substrate or effector) increases binding of ligands to other sites of the protein it is called positive cooperativity, and the opposite regulation is called negative cooperativity. Most allosteric proteins function as points of feedback (inhibition by a downstream product) or feed-forward (activation by a downstream product or parallel reaction) regulation. These metabolites can influence either Km or vmax of the catalyzed reaction.
Threonine deaminase (TD) is a tetrameric enzyme that catalyzes the first step of isoleucine biosynthesis, the conversion of threonine to 2-ketobutyrate. The rate of this reaction proceeds in a sigmoidal manner which can shift depending on effector concentrations. Its activity is decreased by isoleucine, the end product of the reaction, and increased by valine, the end product of a parallel pathway. An experiment was carried out using TDS86G and TDL447,451,454A mutants. The first mutant is catalytically inactive and the second one does not bind any regulatory ligand. Specific activity of TD was measured
Table 1. Specific activity of TD in the presence and absence of isoleucine.
|TD Tetramer Type
Used in Assay
|Active Site Mutant
|Equimolar Mixture of Mutants (hybird tetramer)
|Half of Maximum
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