Studies of thiamin diphosphate binding to the yeast apotransketolase

Previously it was shown that the binding of thiamin diphosphate proceeds through two steps: fast primary binding and the subsequent slow conformational transition of the apoprotein. In the presence of Ca2+, the coenzyme binding occurs with negative cooperativity—owing to the increased rate of the reverse conformational transfer in one of the active centers after completion of ThDP binding at both active centers. There are three viewpoints on the enzyme behavior upon replacement of Ca2+ with Mg2+: (a) negative cooperativity between the two centers is retained; (b) turns positive; (c) totally disappears. In this work, a comparative investigation of the interaction between ThDP and apotransketolase was undertaken and the negative cooperativity between the two centers in the presence of Mg2+, just as in the presence of Ca2+ was demonstrated—albeit with the former cation it was somewhat less pronounced. The negative cooperativity with Mg2+, just as with Ca2+, was caused by an increase in the rate of reverse conformational transfer after the ThDP binding completion in both active centers.