Determination of Constants of Substrate Primary Binding with Baker's Yeast Transketolase by Kinetic Modelling

V. A. Selivanov,¹ L. E. Meshalkina,¹ M. V. Kovina,¹ and G. A. Kochetov^1,2

¹Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119889 Russia; fax: (7-095) 939-31-81; E-mail: kochetov@bac.genebee.msu.su

²To whom correspondence should be addressed.

Submitted November 29, 1996.

A kinetic model of bisubstrate reaction catalyzed by baker's yeast transketolase is proposed. The model considers individual stages of substrate reversible primary binding. The model corresponds to the observed kinetics of product accumulation within a wide range of initial substrate concentrations. Kinetic parameters for the best simulation of the experimental data are defined. The equilibrium constants of the primary binding of both the initial and produced ketose and also of the initial aldose were unequivocally determined by varying the initial substrate concentrations. The dissociation constants of the primary enzyme--substrate complex for the initial ketose (xylulose 5-phosphate) and the reaction product (sedoheptulose 7-phosphate) were found to differ by more than by two orders of magnitude. The result is discussed in the context of the hypothesis of flip-flop functioning of the transketolase active sites.

KEY WORDS: transketolase, kinetic model, substrate primary binding.

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