text: The entrance to the active site for this enzyme is made up mainly of several arginine, histidine, serine, and aspartate side-chains, with a glutamate side-chain playing a secondary role. These side-chains, to be specific Arg359, Arg528, His469, and Ser386, are conserved within each transketolase enzyme and interact with the phosphate group of the donor and acceptor substrates. Because the substrate channel is so narrow, the donor and acceptor substrates cannot be bound simultaneously. Also, the substrates conform into a slightly extended form upon binding in the active site to accommodate this narrow channel.
  Although this enzyme is able to bind numerous types of substrates, such as phosphorylated and nonphosphorylated monosaccharides including the keto and aldosugars fructose, ribose, etc., it has a high specificity for the stereoconfiguration of the hydroxyl groups of the sugars.  These hydroxyl groups at C-3 and C-4 of the ketose donor must be in the D-threo configuration in order to correctly correspond to the C-1 and C-2 positions on the aldose acceptor.  Also they stabilize the substrate in the active site by interacting with the Asp477, His30, and His263 residues.  Disruption of this configuration, both the placement of hydroxyl groups or their stereochemistry, would consequently alter the H-bonding between the residues and substrates thus causing a lower affinity for the substrates.
  In the first half of this pathway, His263 is used to effectively abstract the C3 hydroxyl proton, which thus allows a 2-carbon segment to be cleaved from fructose 6-phosphate. The cofactor necessary for this step to occur is thiamin pyrophosphate (TPP).  The binding of TPP to the enzyme incurs no major conformational change to the enzyme; instead, the enzyme has two flexible loops at the active site that make TPP accessible and binding possible. Thus, this allows the active site to have a "closed" conformation rather than a large conformational change. Later in the pathway, His263 is used as a proton donor for the substrate acceptor-TPP complex, which can then generate erythrose-4-phosphate.
  The histidine and aspartate side-chains are used to effectively stabilize the substrate within the active site and also participate in deprotonation of the substrate.  To be specific, the His 263 and His30 side-chains form hydrogen bonds to the aldehyde end of the substrate, which is deepest into the substrate channel, and Asp477 forms hydrogen bonds with the alpha hydroxyl group on the substrate, where it works to effectively bind the substrate and check for proper stereochemistry.  It is also thought that Asp477 could have important catalytic effects because of its orientation in the middle of the active site and its interactions with the alpha hydroxyl group of the substrate.  Glu418, which is located in the deepest region of the active site, plays a critical role in stabilizing the TPP cofactor.  To be specific, it is involved in the cofactor-assisted proton abstraction from the substrate molecule.
  The phosphate group of the substrate also plays an important role in stabilizing the substrate upon its entrance into the active site.  The tight ionic and polar interactions between this phosphate group and the residues Arg359, Arg528, His469, and Ser386 collectively work to stabilize the substrate by forming H-bonds to the oxygen atoms of the phosphate. The ionic nature is found in the salt bridge formed from Arg359 to the phosphate group.