Mechanistic roles of Thr134, Tyr160, and Lys 164 in the reaction catalyzedby dTDP-glucose 4,6-dehydratase

Escherichia coli dTDP-glucose 4,6-dehydratase and UDP-galactose 4-epimerase are members of the short-chain dehydrogenase/reductase SDR family. A highl y conserved triad consisting of Ser/Thr, Tyr, and Lys is present in the act ive sites of these enzymes as well in other SDR proteins. Ser124, Tyr149, a nd Lys153 in the active site of UDP-galactose 4-epimerase are located in si milar positions as the corresponding Thr134, Tyr160, and Lys164, in the act ive site of dTDP-glucose 4,6-dehydratase. The role of these residues in the first hydride transfer step of the dTDP-glucose 4,6-dehydratase mechanism has been studied by mutagenesis and steady-state kinetic analysis. In all m utants except T134S, the k(cat) values are more than 2 orders of magnitude lower than of wild-type enzyme. The substrate analogue, dTDP-xylose, was us ed to investigate the effects of the mutations on rate of the first hydride transfer step. The first step becomes significantly rate limiting upon mut ation of Tyr160 to Phe and only partly rate limiting in the reaction cataly zed by K164M and T134A dehydratases. The pH dependence of k(cat), the stead y-state NADH level, and the fraction of NADH formed with saturating dTDP-xy lose show shifts in the pKa assigned to Tyr160 to more basic values by muta tion of Lys164 and Thr134. The pKa of Tyr160, as determined by the pH depen dence of NADH formation by dTDP-xylose, is 6.41. Lys164 and Thr134 are beli eved to play important roles in the stabilization of the anion of Tyr160 in a fashion similar to the roles of the corresponding residues in UDP-galact ose 4-epimerase, which facilitate the ionization of Tyr149 in that enzyme [ Liu, Y., et al. (1997) Biochemistry 35, 10675-10684]. Tyr160 is presumably the base for the first hydride transfer step, while Thr134 may relay a prot on from the sugar to Tyr160.