W0220

Studies on the Structure and Function of Thiamin Pyrophosphokinase. Jingyuan Liu, David E. Timm, Robert A. Harris, and Thomas D. Hurley, Dept. of Biochemistry and Molecular Biology, Indiana Univ. School of Medicine, Indianapolis, IN 46202-5122.

Thiamin pyrophosphokinase (TPK) converts thiamin into its active form thiamin pyrophophsphate (TPP) by transferring a pyrophosphate group from ATP. TPP is a prosthetic group for four metabolically important enzymes: branched chain α-keto acid dehydrogenase, pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and transketolase. Thiamin deficiency results in Wernike-Korsakof Syndrome (WKS) and beriberi disease. Thiamin deficiency is also a frequent complication of alcoholism. In this study mouse thiamin pyrophosphokinase (mTPK), which has more than 90% sequence similarity to human TPK, was expressed in E. coli and purified to homogeneity. Activity assays of the purified enzyme were performed with a coupled enzyme system. Inorganic phosphate, which has been used in most previous assay buffers for the enzyme, inhibits activity of the recombinant enzyme. Ethanol, which has been reported to inhibit the native enzyme, is without effect upon the recombinant enzyme. Crystal structures of mTPK complexed with the inhibitor pyrithiamin and the product TPP were determined at 1.95 Å and 1.7 Å, respectively. Both pyrithiamin and TPP were found located in the thiamin binding pocket. Compared with the high-energy “V” conformation, which is adopted by the coenzyme TPP in enzymes that utilize TPP as a cofactor, thiamin, pyrithiamin and TPP in mTPK adopt the low-energy “F” conformation.