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. Mechanism 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. Role in disease Transketolase activity is decreased in deficiency of thiamine, which in general is due to malnutrition. Several diseases are associated with thiamine deficiency, including beriberi, biotin-thiamine-responsive basal ganglia disease (BTBGD), Wernicke–Korsakoff syndrome, and others (see thiamine for a comprehensive listing). In Wernicke–Korsakoff syndrome, while no mutations could be demonstrated, there is an indication that thiamine deficiency leads to Wernicke–Korsakoff syndrome only in those whose transketolase has a reduced affinity for thiamine. In this way, the activity of transketolase is greatly hindered, and, as a consequence, the entire pentose phosphate pathway is inhibited. Diagnostic use Red blood cell transketolase activity is reduced in deficiency of thiamine (vitamin B1), and may be used in the diagnosis of Wernicke encephalopathy and other B1-deficiency syndromes if the diagnosis is in doubt. Apart from the baseline enzyme activity (which may be normal even in deficiency states), acceleration of enzyme activity after the addition of thiamine pyrophosphate may be diagnostic of thiamine deficiency (0-15% normal, 15-25% deficiency, >25% severe deficiency). References Photosynthesis EC 2.2.1