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Site-specific mutagenesis of Drosophila alcohol dehydrogenase: evidence for involvement of tyrosine-152 and lysine-156 in catalysis.

Publication ,  Journal Article
Chen, Z; Jiang, JC; Lin, ZG; Lee, WR; Baker, ME; Chang, SH
Published in: Biochemistry
April 6, 1993

Amino acid sequence comparisons reveal that tyrosine-152 and lysine-156 of Drosophila alcohol dehydrogenase (ADH) are conserved in homologous dehydrogenases, suggesting that these residues are important in catalysis. To test this hypothesis, we used site-directed mutagenesis to substitute tyrosine-152 with phenylalanine, histidine, or glutamic acid or to substitute lysine-156 with isoleucine. All of these mutants are catalytically inactive. Two mutants were active: A cysteine mutation of tyrosine-152 has 0.25% of wild-type ADH activity, while an arginine substitution of lysine-156 retains 2.2% of wild-type ADH activity. Kinetic analysis shows that the cysteine mutant increases Km(ethanol) 56-fold and Km(propan-2-ol) 100-fold, while Km(NAD) values are essentially unaltered. The arginine mutant also shows the significant enlargement of Km(ethanol), but not of Km(NAD). Furthermore, the cysteine mutant and arginine mutant have different substrate specificity and behave differently on competitive inhibition than wild-type ADH. These results suggest that both tyrosine-152 and lysine-156 have essential roles in catalysis by Drosophila ADH.

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Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

April 6, 1993

Volume

32

Issue

13

Start / End Page

3342 / 3346

Location

United States

Related Subject Headings

  • Tyrosine
  • Substrate Specificity
  • Recombinant Proteins
  • Mutagenesis, Site-Directed
  • Molecular Sequence Data
  • Kinetics
  • Hydrogen-Ion Concentration
  • Ethanol
  • Drosophila melanogaster
  • Catalysis
 

Citation

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Chen, Z., Jiang, J. C., Lin, Z. G., Lee, W. R., Baker, M. E., & Chang, S. H. (1993). Site-specific mutagenesis of Drosophila alcohol dehydrogenase: evidence for involvement of tyrosine-152 and lysine-156 in catalysis. Biochemistry, 32(13), 3342–3346. https://doi.org/10.1021/bi00064a017
Chen, Z., J. C. Jiang, Z. G. Lin, W. R. Lee, M. E. Baker, and S. H. Chang. “Site-specific mutagenesis of Drosophila alcohol dehydrogenase: evidence for involvement of tyrosine-152 and lysine-156 in catalysis.Biochemistry 32, no. 13 (April 6, 1993): 3342–46. https://doi.org/10.1021/bi00064a017.
Chen Z, Jiang JC, Lin ZG, Lee WR, Baker ME, Chang SH. Site-specific mutagenesis of Drosophila alcohol dehydrogenase: evidence for involvement of tyrosine-152 and lysine-156 in catalysis. Biochemistry. 1993 Apr 6;32(13):3342–6.
Chen, Z., et al. “Site-specific mutagenesis of Drosophila alcohol dehydrogenase: evidence for involvement of tyrosine-152 and lysine-156 in catalysis.Biochemistry, vol. 32, no. 13, Apr. 1993, pp. 3342–46. Pubmed, doi:10.1021/bi00064a017.
Chen Z, Jiang JC, Lin ZG, Lee WR, Baker ME, Chang SH. Site-specific mutagenesis of Drosophila alcohol dehydrogenase: evidence for involvement of tyrosine-152 and lysine-156 in catalysis. Biochemistry. 1993 Apr 6;32(13):3342–3346.
Journal cover image

Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

April 6, 1993

Volume

32

Issue

13

Start / End Page

3342 / 3346

Location

United States

Related Subject Headings

  • Tyrosine
  • Substrate Specificity
  • Recombinant Proteins
  • Mutagenesis, Site-Directed
  • Molecular Sequence Data
  • Kinetics
  • Hydrogen-Ion Concentration
  • Ethanol
  • Drosophila melanogaster
  • Catalysis