Transketolase kinetics. The slow reconstitution of the holoenzyme is due to rate-limiting dimerization of the subunits.

Reassociation of the pyruvate dehydrogenase complex from Escherichia coli: kinetic measurements and binding studies by resonance energy transfer

“…Upon mixing of the components a considerable lag period is observed. Similar behavior has been reported for cytoplasmic pyruvate decarboxylase and transketolase from yeast (Hübner et al, 1978;Egan & Sable, 1981).…”

Inhibition of pyruvate decarboxylase from Z. mobilis by novel analogues of thiamine pyrophosphate: investigating pyrophosphate mimics

“…TPP itself has been reported to show negative cooperativity in its binding to transketolase. 8 In support of the second explanation, we note that the apparent rate constant for the slower of the two stages also seems to be dependent on concentration of inhibitor.…”

Structural basis for the magnesium-dependent activation of transketolase from Chlamydomonas reinhardtii

“…Indeed, short exposure of the CrTKTPP to Mg 2+ ions during activity assay (2 min) is not sufficient to allow the enzyme to adopt suited conformation for optimal catalysis. This agrees with previous studies that analyzed the rate of transition from the apo-form of yeast TK to the fully active holo-form, demonstrating that the long time needed to reach the maximal activity is strongly decreased upon pre-incubation of the protein with both TPP and Mg 2+ [8,36]. The formation of fully reconstituted TPP-dependent enzymes can be monitored by the appearance of UV and ICD signals upon TPP binding [39].…”

“…). In agreement with what reported about the activation of yeast TK[36], when CrTKTPP was not pre-incubated in the presence of Mg 2+ the enzyme exhibited a lower activity, even if exposed to saturating concentrations of divalent cations (i.e. Mg 2+ and Ca 2+ ) during the activity assays.As the coupled assay used to measure CrTK activity was conducted at high concentrations of Mg2+ and Ca 2+ , we have also determined the dependence of CrTKTPP and CrTKTPP/Mg activity as a function of Mg 2+ concentration in the assay buffer deprived of calcium ions.…”