Spectroscopic studies of inhibited alcohol dehydrogenase from Thermoanaerobacter brockii: Proposed structure for the catalytic intermediate state

Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) catalyzes the reve rsible oxidation of secondary alcohols to the corresponding ketones using N ADP(+) as the cofactor. The active site of the enzyme contains a zinc ion t hat is tetrahedrally coordinated by four protein residues. The enzymatic re action leads to the formation of a ternary enzyme-cofactor-substrate comple x; and catalytic hydride ion transfer is believed to take place directly be tween the substrate and cofactor at the ternary complex. Although crystallo graphic data of TbADH and other alcohol dehydrogenases as well as their com plexes are available, their mode of action remains to be determined. It is firmly established that the zinc ion is essential for catalysis. However, t here is no clear agreement about the coordination environment of the metal ion and the competent reaction intermediates during catalysis. We used a co mbination of X-ray absorption, circular dichroism (CD), and fluorescence sp ectroscopy, together with structural analysis and modeling studies, to inve stigate the ternary complexes of TbADH that are bound to a transition-state analogue inhibitor. Our structural and spectroscopic studies indicated tha t the coordination sphere of the catalytic zinc site in TbADH undergoes con formational changes when it binds the inhibitor and forms a pentacoordinate d complex at the zinc ion. These studies provide the first active site stru cture of bacterial ADH bound to a substrate analogue. Here, we suggest the active site structure of the central intermediate complex and, more specifi cally, propose the substrate-binelin. (C) site in TbADH.