Site-directed mutagenesis of glycine-14 and two "critical" cysteinyl residues in Drosophila alcohol dehydrogenase.
Three amino acid residues (glycine-14, cysteine-135, and cysteine-218) previously speculated to be important for the structure and function of Drosophila melanogaster alcohol dehydrogenase have been investigated by using site-directed mutagenesis followed by kinetic analysis and chemical modification. Mutating glycine-14 to valine (G14V) virtually inactivates Drosophila ADH, and substitution of alanine at this position (G14A) causes a 31% decrease in activity. Thermal denaturation and kinetic and inhibition studies further demonstrate 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 the correct conformation in the NAD binding domain. On the other hand, changing of cysteine-135, -218, or both to alanine (C135A, C218A, and C135A/C218A) causes no decrease in the catalytic activity of the enzyme, indicating that neither of the cysteinyl residues is essential for catalysis. C135A and wild-type enzyme are both inactivated by DTNB. In contrast, C218A and C135A/C218A are unaffected by DTNB treatment. DTNB modification of cysteine-218 can be prevented by the substrates NAD and 2-propanol, suggesting 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 2-propanol and/or NAD, suggesting a conformational change induced by binding of these substrates.
Chen
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Department of Biochemistry, Louisiana State University, Baton Rouge 70803.
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Shirley
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Lee
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Chang
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D000426:Alcohol Dehydrogenase / Q000037:antagonists & inhibitors / Q000235:genetics*; D000818:Animals; D003545:Cysteine / Q000235:genetics*; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics*; D004789:Enzyme Activation / Q000187:drug effects; D015870:Gene Expression; D005998:Glycine / Q000235:genetics*; D007700:Kinetics; D009154:Mutation; D016133:Polymerase Chain Reaction; D011489:Protein Denaturation
D004247:DNA; D004228:Dithionitrobenzoic Acid; D000426:Alcohol Dehydrogenase; D003545:Cysteine; D005998:Glycine