Mechanistic and Structural Characterization of Thiamine Diphosphate Dependent Enzyme Transketolases from Human and E.coli

Abstract

Thiamine diphosphate (ThDP) dependent enzymes are essential in the metabolism of biological systems, and many of these enzymes are extensively used in the biosynthetic of valuable compounds. In this regard, a comprehensive study of the catalytic mechanism of this family of enzymes are demanding, especially high resolution structural information. In this thesis, we present the mechanistic study of transketolase, one representative ThDP dependent enzyme, from several different aspects. First we show that a low barrier hydrogen bond (LBHB) is present in the crystal structure of human transketolase, and the function of this unusual hydrogen bond within the proton tunnel is studied, this gives insight to the long debated topic of the function of LBHB in enzyme catalysis. Then, the function of the phosphate group on native substrates are shown to be crucial for the stabilization of the substrate-cofactor intermediate, and by supplying exogenous phosphite dianion, the reaction can be accelerated by several fold. In addition, a ring flipping of histidine in E.coli transketolase and a structural plasticity of the active site in human transketolase have been studied.