Site-directed mutagenesis of glycine-14 and two "critical" cysteinyl residues in Drosophila alcohol dehydrogenase.

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Chen, Z; Lu, L; Shirley, M; Lee, WR; Chang, SH

Published Date

  • February 6, 1990

Published In

Volume / Issue

  • 29 / 5

Start / End Page

  • 1112 - 1118

PubMed ID

  • 2108721

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi00457a003

Language

  • eng

Conference Location

  • United States