The overall goal of these studies is to learn more about the detailed mechanism of horse liver alcohol dehydrogenase, in particular about conformational changes that occur when coenzymes bind and when ternary complexes interconvert. The rates of the conformational changes will be determined with various spectral probes using rapid reaction techniques. Since modification of lysine residue 228 increases the activity up to 10-fold, apparently by altering the rate at which enzyme changes conformation, the mechanism of the activation and the rates of the conformational change will be determined. The roles of functional groups at the active site will be elucidated by chemical modification and pH dependency studies. The equilibrium position for the interconversion of ternary complexes (""""""""internal equilibrium""""""""), which is a measure of the """"""""efficiency"""""""" of the enzyme will be determined. The pH dependencies, the effects of variation of redox potential of the reactants, and the propensity to bind in """"""""non-productive"""""""" modes will be characterized. The size and shape of the substrate binding pocket will be defined in relation to the three-dimensional structure of the protein. Based on knowledge of the structure and mechanism of the enzyme, specific activators will be synthesized and evaluated. One type of activator is a coenzyme analog with functional groups that can methylate the amino group of lysine residue 228. A second type of activator is one that participates in a coupled exchange reaction and oxidizes the enzyme NADH complex, thereby by-passing the rate-limiting dissociation of coenzyme. These activators should be useful for defining the rate-limiting role of alcohol dehydrogenase in ethanol metabolism and eventually for the treatment of acute and chronic alcoholism.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA000279-20
Application #
2042946
Study Section
Biochemistry, Physiology and Medicine Subcommittee (ALCB)
Project Start
1979-06-01
Project End
1993-11-30
Budget Start
1992-09-01
Budget End
1993-11-30
Support Year
20
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Iowa
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041294109
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Kim, Keehyuk; Plapp, Bryce V (2017) Inversion of substrate stereoselectivity of horse liver alcohol dehydrogenase by substitutions of Ser-48 and Phe-93. Chem Biol Interact 276:77-87
Plapp, Bryce V; Savarimuthu, Baskar Raj; Ferraro, Daniel J et al. (2017) Horse Liver Alcohol Dehydrogenase: Zinc Coordination and Catalysis. Biochemistry 56:3632-3646
Plapp, Bryce V; Leidal, Kevin G; Murch, Bruce P et al. (2015) Contribution of liver alcohol dehydrogenase to metabolism of alcohols in rats. Chem Biol Interact 234:85-95
Yahashiri, Atsushi; Rubach, Jon K; Plapp, Bryce V (2014) Effects of cavities at the nicotinamide binding site of liver alcohol dehydrogenase on structure, dynamics and catalysis. Biochemistry 53:881-94
Kovaleva, Elena G; Plapp, Bryce V (2005) Deprotonation of the horse liver alcohol dehydrogenase-NAD+ complex controls formation of the ternary complexes. Biochemistry 44:12797-808