Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Biological Sciences

First Advisor

Simon H. Chang


Alcohol dehydrogenase (ADH, alcohol:NAD oxidoreductase, EC1.1.1.1) from Drosophila melanogaster (DmADH) is unique among alcohol dehydrogenases in metal ion requirement, substrate preference, and stereospecificity of hydride transfer. In order to study this unique enzyme, the first part of this dissertation reports the construction and expression of DmADH cDNA in E. coli hosts. The second part of the dissertation shows the structural function of glycine-14 in putative NAD-binding domain of DmADH. The comparison of DmADH to horse liver ADH suggests that glycine-14 in DmADH is located at the first position of a "conserved sequence" which maintains the tight turn structure positioned in the NAD-binding domain. Mutating glycine 14 to valine virtually inactivates DmADH and alanine substitution causes a decrease in activity. Thermal denaturation, kinetic, and inhibition studies confirm that replacing glycine-14 with either alanine or valine leads to structural changes in the NAD-binding domain. These results provide direct evidence for the role played by glycine-14 in maintaining correct conformation in the NAD-binding domain. The third part of the dissertation concerns two cysteinyl residues. DmADH is sensitive to DTNB which modifies one of the two cysteinyl residues in the enzyme. In order to identify the reactive cysteinyl residue and investigate its functional significance, one or both of the two cysteinyl residues has been changed to alanine (CA135, CA218 and CA135/CA218). None of the mutants show decreased specific activity relative to wild-type, indicating that neither of the cysteinyl residues is essential for catalysis. CA135 wild-type enzyme are both inactivated by DTNB, but CA218 and CA135/CA218 are unaffected by DTNB treatment, suggesting that DTNB introduces a steric interference at cysteine-218. DTNB modification of cysteine can be prevented by the substrates, NAD and 2-propanol, indicating that cysteine-218 may be in the vicinity of the active site. Cysteine-135 which is normally insensitive to DTNB becomes accessible in the presence of isopropanol and NAD, suggesting a conformational change induced by binding to these substrates.