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

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.

[1]  S. Benner,et al.  A stereochemical imperative in dehydrogenases: new data and criteria for evaluating function-based theories in bioorganic chemistry , 1985 .

[2]  L. Sawyer,et al.  Preliminary X-ray crystallographic studies on alcohol dehydrogenase from Drosophila. , 1992, Journal of molecular biology.

[3]  A. Place,et al.  Alcohol dehydrogenase gene of Drosophila melanogaster: relationship of intervening sequences to functional domains in the protein. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[4]  H. Jörnvall,et al.  The primary structure of alcohol dehydrogenase from Drosophila lebanonensis. Extensive variation within insect 'short-chain' alcohol dehydrogenase lacking zinc. , 1989, European journal of biochemistry.

[5]  T. Kunkel.  Rapid and efficient site-specific mutagenesis without phenotypic selection. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[6]  P. White,et al.  Tyr-179 and Lys-183 are essential for enzymatic activity of 11 beta-hydroxysteroid dehydrogenase. , 1992, Biochemical and biophysical research communications.

[7]  D. Thatcher.  The complete amino acid sequence of three alcohol dehydrogenase alleloenzymes (AdhN-11, AdhS and AdhUF) from the fruitfly Drosophila melanogaster. , 1980, The Biochemical journal.

[8]  R. Hovik,et al.  Drosophila melanogaster alcohol dehydrogenase: Substrate sterospecificity of the AdhF alleloenzyme , 1984 .

[9]  K. Dalziel,et al.  Initial Steady State Velocities in the Evaluation of Enzyme-Coenzyme-Substrate Reaction Mechanisms. , 1957 .

[10]  R. Gonzàlez-Duarte,et al.  Biochemical properties of alcohol dehydrogenase from Drosophila lebanonensis. , 1986, The Biochemical journal.

[11]  K. Dalziel.  Kinetic studies of liver alcohol dehydrogenase. , 1962, The Biochemical journal.

[12]  M. Baker.  Sequence similarity between Pseudomonas dihydrodiol dehydrogenase, part of the gene cluster that metabolizes polychlorinated biphenyls, and dehydrogenases involved in metabolism of ribitol and glucitol and synthesis of antibiotics and 17 beta-oestradiol, testosterone and corticosterone. , 1990, The Biochemical journal.

[13]  M. Baker,et al.  A common ancestor for human placental 17β‐hydroxysteroid dehydrogenase, Streptomyces coelicolor actIII protein, and Drosophila melanogaster alcohol dehydrogenase , 1990, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[14]  M Krook,et al.  Characteristics of short-chain alcohol dehydrogenases and related enzymes. , 1991, European journal of biochemistry.

[15]  J. Winberg,et al.  Kinetic interpretations of active site topologies and residue exchanges in Drosophila alcohol dehydrogenases. , 1992, The International journal of biochemistry.

[16]  P. White,et al.  The human gene for 11 beta-hydroxysteroid dehydrogenase. Structure, tissue distribution, and chromosomal localization. , 1991, The Journal of biological chemistry.

[17]  Spectra of molecular changes induced in DNA of Drosophila spermatozoa by 1-ethyl-1-nitrosourea and X-rays. , 1988 .

[18]  Z. Chen,et al.  Site-directed mutagenesis of glycine-14 and two "critical" cysteinyl residues in Drosophila alcohol dehydrogenase. , 1990, Biochemistry.

[19]  S. F. Baron,et al.  Cloning, sequencing, and expression of the gene coding for bile acid 7 alpha-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708 , 1991, Journal of bacteriology.

[20]  H. Tai,et al.  Site-directed mutagenesis of the conserved tyrosine 151 of human placental NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase yields a catalytically inactive enzyme. , 1991, Biochemical and biophysical research communications.

[21]  D. Thatcher,et al.  Alcohol dehydrogenase from the fruitfly Drosophila melanogaster. Substrate specificity of the alleloenzymes AdhS and AdhUF. , 1982, Biochimica et biophysica acta.

[22]  Z. Chen,et al.  Role of aspartic acid 38 in the cofactor specificity of Drosophila alcohol dehydrogenase. , 1991, European journal of biochemistry.

[23]  D. Ghosh,et al.  Three-dimensional structure of holo 3 alpha,20 beta-hydroxysteroid dehydrogenase: a member of a short-chain dehydrogenase family. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[24]  J. Winberg,et al.  The AdhS alleloenzyme of alcohol dehydrogenase from Drosophila melanogaster. Variation of kinetic parameters with pH. , 1988, The Biochemical journal.

[25]  R. Wierenga,et al.  INTERACTION OF PYROPHOSPHATE MOIETIES WITH ALPHA-HELIXES IN DINUCLEOTIDE BINDING-PROTEINS , 1985 .

[26]  Michael E. Baker,et al.  Genealogy of regulation of human sex and adrenal function, prostaglandin action, snapdragon and petunia flower colors, antibiotics, and nitrogen fixation: functional diversity from two ancestral dehydrogenases , 1991, Steroids.

[27]  A. Leo,et al.  Partition coefficients and their uses , 1971 .