The overall goal of this project is to identify specific inhibitors and substrates for the human beta, gamma, and sigma alcohol dehydrogenase (ADH) isoenzymes. Dietary and biogenic alcohols and aldehydes are primarily metabolized by the NAD(H) dependent alcohol dehydrogenase isoenzymes within the cell. There has been considerable debate concerning the involvement of alcohol dehydrogenase isoenzymes in the metabolism of ethanol. The primary reason for this controversy is the lack of specific inhibitors or substrates with which to evaluate their activities in vivo or in vitro. We propose to utilize a structure-based approach toward the identification of specific inhibitors and substrates for the human beta, gamma, and sigma ADH isoenzymes. We have recently solved the three-dimensional structures of all three of these ADH isoenzymes. We have finished the structure refinement of the human beta forms and are continuing the refinement of the gamma and sigma ADH structures. We propose to use these structures to identify inhibitors based on their active site characteristics with the recently developed computer programs DOCK and AUTODOCK. The three- dimensional structures of these isoenzymes will be used in docking experiments to identify specific substrates and inhibitors for these important ADH isoenzymes. Our initial studies will characterize the determinants of specific binding to the respective isoenzymes using a variety of in vitro methods, such as steady-state and stopped-flow kinetic measurements, as well as X-ray crystallography of enzyme-inhibitor complexes. A long term goal of this proposal is to develop stably transformed cell lines expressing one or more of these isoenzymes for evaluation of in vivo inhibitors and toxicological properties of potential inhibitors and substrates. These studies should contribute to the basic understanding of the involvement of these human ADH isoenzymes in the overall rate of ethanol metabolism and may identify important intracellular substrates for these ADH isoenzymes.

National Institute of Health (NIH)
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
Application #
Study Section
Biochemistry, Physiology and Medicine Subcommittee (ALCB)
Program Officer
Isaki, Leslie
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Indiana University-Purdue University at Indianapolis
Schools of Medicine
United States
Zip Code
Gibbons, Brian J; Hurley, Thomas D (2004) Structure of three class I human alcohol dehydrogenases complexed with isoenzyme specific formamide inhibitors. Biochemistry 43:12555-62
Sanghani, Paresh C; Robinson, Howard; Bennett-Lovsey, Riccardo et al. (2003) Structure-function relationships in human Class III alcohol dehydrogenase (formaldehyde dehydrogenase). Chem Biol Interact 143-144:195-200
Sanghani, Paresh C; Bosron, William F; Hurley, Thomas D (2002) Human glutathione-dependent formaldehyde dehydrogenase. Structural changes associated with ternary complex formation. Biochemistry 41:15189-94
Sanghani, Paresh C; Robinson, Howard; Bosron, William F et al. (2002) Human glutathione-dependent formaldehyde dehydrogenase. Structures of apo, binary, and inhibitory ternary complexes. Biochemistry 41:10778-86
Niederhut, M S; Gibbons, B J; Perez-Miller, S et al. (2001) Three-dimensional structures of the three human class I alcohol dehydrogenases. Protein Sci 10:697-706
Xie, P T; Hurley, T D (1999) Methionine-141 directly influences the binding of 4-methylpyrazole in human sigma sigma alcohol dehydrogenase. Protein Sci 8:2639-44
Hurley, T D; Steinmetz, C G; Weiner, H (1999) Three-dimensional structure of mitochondrial aldehyde dehydrogenase. Mechanistic implications. Adv Exp Med Biol 463:15-25
Yang, Z N; Bosron, W F; Hurley, T D (1997) Structure of human chi chi alcohol dehydrogenase: a glutathione-dependent formaldehyde dehydrogenase. J Mol Biol 265:330-43
Xie, P; Parsons, S H; Speckhard, D C et al. (1997) X-ray structure of human class IV sigmasigma alcohol dehydrogenase. Structural basis for substrate specificity. J Biol Chem 272:18558-63
Hurley, T D; Steinmetz, C G; Xie, P et al. (1997) Three-dimensional structures of human alcohol dehydrogenase isoenzymes reveal the molecular basis for their functional diversity. Adv Exp Med Biol 414:291-302