The overall goal of this research project is to determine the relationships between the catalytic function and three-dimensional structure of human alcohol dehydrogenases (ADH). There are as many as nine different genetically encoded subunits of alcohol dehydrogenase which combine to form more than 20 different active dimeric isoenzymes. We have shown that the kinetic properties of these isoenzymes differ widely. In order to investigate structure-function relationships among these isoenzymes, we devised methods to express recombinant human alcohol dehydrogenases in E. coli and purify them to homogeneity. Recombinant beta1/beta1 was crystallized and its X-ray structure (the first of a human alcohol dehydrogenase) reveals differences from the horse liver enzyme in the coenzyme binding site, which explains the tighter binding of coenzyme to beta1/beta1 versus the horse liver enzyme. We will determine the X-ray structures of isoenzymes with different catalytic properties from beta1/beta1, for example, alpha/alpha, pi/pia, chi/chi, and the eta-like stomach enzyme. We are particularly interested in determining X-ray structures in enzyme substrate complexes which will elucidate the unique catalytic properties of ADH isoenzymes, for example, the crystallization of chichi with s-formylglutathione and NAD+ might yield important information regarding the mechanism of chichi as a formaldehyde dehydrogenase. We have created variants of beta1/beta1 by site-directed mutagenesis, expressed the variant enzymes in E. coli, and purified them to homogeneity. Using these methods we will examine structure-function relationships for the coenzyme and alcohol binding sites for ADH. Based on known X-ray structures, we will make specific mutations in these sites and examine the steady-state and stopped-flow kinetic properties of these mutants to establish the functional roles of these amino acids. We will use intrinsic Trp fluorescence of beta1/beta1 or fluorescence of mutants with Trp residues inserted or deleted in specific regions to probe the substrate and inhibitor specificity for the substrate-induced conformational change in the apo- versus NAD+ trifluoroethanol-enzyme complexes. The enzymatic properties of ADH play a key role in determining the pharmacokinetics of ethanol elimination in man. These studies should contribute to the basic understanding of the relative contribution of the various isoenzymes of human ADH to ethanol metabolism. They should also contribute to the basic biochemical knowledge about the structure and catalytic mechanisms of the human alcohol dehydrogenases.
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