Half-of-the-sites reactivity of transketolase from Saccharomyces cerevisiae

Cleavage by yeast transketolase of the donor substrate, d-xylulose 5-phosphate, in the absence of the acceptor substrate was studied using stopped-flow spectrophotometry. One mole of the substrate was shown to be cleaved in the prestationary phase, leading to the formation of one mole of the reaction product per mole enzyme, which has two active centers. This observation indicates that only one out of the two active centers functions (i.e., binds and cleaves the substrate) at a time. Such half-of-the-sites reactivity of transketolase conforms well with our understanding, proposed previously, that the active centers of the enzyme operate in sequence (in phase opposition): the cleavage of a ketose within one center (first phase of the transketolase reaction) is paralleled by its formation in the other center (glycolaldehyde residue is condensed with the acceptor substrate, and the second stage of the transketolase reaction is thereby completed) [M.V. Kovina, G.A. Kochetov, FEBS Lett. 440 (1998) 81–84].