The long-term objective of this research project is to determine the three- dimensional structure of the multiple molecular forms of the two principal enzymes involved in the metabolism of ethanol; alcohol dehydrogenase and aldehyde dehydrogenase. The hypothesis is that knowledge of the three- dimensional structure of the substrate binding sites of the various isoenzymes will allow the prediction of their substrate specificities as well as their contributions to the metabolism of ethanol. Specifically, I will crystallize and determine the three-dimensional structures of the human beta1beta1, alphaalpha, pipi, and chichi alcohol dehydrogenase enzymes complexed with alcohol substrates and the mitochondrial form of aldehyde dehydrogenase complexed with the coenzyme NAD(H). The recombinant proteins, expressed and purified from E. coli, will be used for this purpose to alleviate problems associated with minor contamination of closely related isoenzyme forms. These forms of human alcohol dehydrogenase possess amino acid substitutions within their substrate binding sites and it is the objective of this proposal to evaluate the effects of these substitutions on substrate specificity and maximal catalytic activity. A three-dimensional structure for the mitochondrial form of aldehyde dehydrogenase or closely related enzymes is not available. Accordingly, little is known about the determinants of catalytic activity or substrate specificity for the aldehyde dehydrogenase isoenzymes. The elucidation of its three-dimensional structure by X-ray crystallography will provide the necessary structural model for determining these functional relationships.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Scientist Development Award (K21)
Project #
5K21AA000150-02
Application #
3089028
Study Section
Biochemistry, Physiology and Medicine Subcommittee (ALCB)
Project Start
1992-07-01
Project End
1997-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
2
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Type
Schools of Medicine
DUNS #
005436803
City
Indianapolis
State
IN
Country
United States
Zip Code
46202