W0476

Structural Features Determining Affinity of a Thiamin Dependent Enzyme for Thiamin Diphosphate and Inhibitor. Andrew Brunskill^1,2, Krishnamoorthy Chandrasekhar^{1, 2}, Palaniappa Arjunan^1,2, Lakshminarasimhulu Pasupulati^1,2, Martin Sax², Natalia Nemeria³, Frank Jordan³, William Furey^1,2, ¹Dept. of Pharmacology, Univ. of Pittsburgh, Pittsburgh, PA 15261 & ²Biocrystallography Laboratory, VA Medical Center, Pittsburgh, PA 15240 and ³Dept. of Chemistry, Rutgers Univ., Newark, NJ 07102.

The pyruvate dehydrogenase multienzyme complex (PDHc) plays a central role in cellular energy metabolism. The enzyme consists of multiple copies of three different enzymes that function sequentially, named E1, E2 and E3. The first component from E. coli, thiamin diphosphate (ThDP) dependent pyruvate dehydrogenase (E1p), has been a major focus of research in our laboratory. The inhibitor thiamin thiazolone diphosphate (ThTDP) binds to PDHc E1p ~20,000 times more strongly than does ThDP and replacement of ThDP by ThTDP inactivates the enzyme. The crystal structure of PDHc E1p - ThTDP inhibitor complex at 2.1Å reveals reorganization in the vicinity of the active site, entailing small structural and conformational changes together with the addition of new hydration sites and elimination of some others that are observed when ThDP is present instead. A significant increase in hydrogen bonding is noticed in the active site area in the presence of ThTDP. The study provides insight into the relative binding affinities of thiamin dependent enzymes for ThTDP and ThDP, as well as the role of water in enzymatic catalysis in general.