ASP(477) IS A DETERMINANT OF THE ENANTIOSELECTIVITY IN YEAST TRANSKETOLASE

The conserved residue Asp(477) in yeast transketolase is located in th e substrate channel of the enzyme and forms a hydrogen bond with the C 2-hydroxyl group of the acceptor substrate. The significance of this i nteraction for the recognition of the preferred acceptor substrates, D -alpha-hydroxyaldehydes was investigated by site-directed mutagenesis. In the wild-type enzyme the k(cat)/K-M values are by three to four or ders of magnitude lower for 2-deoxyaldoses or substrates with L-config uration at the C2-atom. In the Asp(477)Ala mutant, the k(cat)/K-M valu es for D-alpha-hydroxyaldehydes are decreased by a thousandfold, while the k(cat)/K-M values for substrates with L-configuration or 2-deoxya ldoses are similar to wild-type enzyme. These results indicate that As p(477) is involved in determining the enantioselectivity of transketol ase. (C) 1998 Federation of European Biochemical Societies.