The interaction of transketolase with its acceptor substrate, ribose 5-phosphate, has been studied. The active centers of the enzyme were shown to be functionally nonequivalent with respect to ribose 5-phosphate binding. Under the conditions where only one out of the two active centers of transketolase is functional, their affinities for ribose 5-phosphate are identical. The phenomenon of nonequivalence becomes apparent when the substrate interacts with one of the two active centers. As a consequence of such interaction, the affinity of the second active center for ribose 5-phosphate decreases. (C) 2007 Elsevier Inc. All rights reserved.