Nilsson U, Hecquet L, Gefflaut T, Guerard C, Schneider G
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
FEBS Lett. 1998 Mar 6;424(1-2):49-52. doi: 10.1016/s0014-5793(98)00136-7.
The conserved residue Asp477 in yeast transketolase is located in the substrate channel of the enzyme and forms a hydrogen bond with the C2-hydroxyl group of the acceptor substrate. The significance of this interaction for the recognition of the preferred acceptor substrates, D-alpha-hydroxyaldehydes was investigated by site-directed mutagenesis. In the wild-type enzyme the kcat/KM values are by three to four orders of magnitude lower for 2-deoxyaldoses or substrates with L-configuration at the C2-atom. In the Asp477 Ala mutant, the kcat/KM values for D-alpha-hydroxyaldehydes are decreased by a thousandfold, while the kcat/KM values for substrates with L-configuration or 2-deoxyaldoses are similar to wild-type enzyme. These results indicate that Asp477 is involved in determining the enantioselectivity of transketolase.
