The catalysis of this mechanism is initiated by the deprotonation of TPP at the thiazolium ring.  This carbanion then binds to the carbonyl of the donor substrate thus cleaving the bond between C-2 and C-3.  This keto fragment remains covalently bound to the C-2 carbon of TPP.  The donor substrate is then released, and the acceptor substrate enters the active site where the fragment, which is bound to the intermediate α-β-dihydroxyethyl thiamin diphosphate, is then transferred to the acceptor.
Experiments have also been conducted that test the effect replacing alanine for the amino acids at the entrance to the active site, Arg359, Arg528, and His469, which interact with the phosphate group of the substrate.  This replacement creates a mutant enzyme with impaired catalytic activity.