Aspartate 46, a second sphere ligand to the catalytic zinc, is essential for activity of yeast alcohol dehydrogenase

Conference · Fri May 01 00:00:00 EDT 1987 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5978039

Ganzhorn, A J; Plapp, B V

The crystal structure of horse liver alcohol dehydrogenase (ADH) shows a hydrogen bond between the imidazole of His-67, a ligand to the active site zinc, and the carboxylate of Asp-49. Both residues are conserved in alcohol dehydrogenases. Directed mutagenesis was used to replace the homologous Asp-46 in ADH I from S. cerevisiae with asparagine. The substitution did not alter the overall structure of the enzyme, as judged by CD measurements, but the removal of a negative charge was evident in electrophoresis, and in the absorption and fluorescence spectra. The mutant and wild-type enzymes had similar zinc contents as determined by atomic absorption spectroscopy. Active site titration and steady state kinetics indicated that binding of coenzymes, substrates and substrate analogs is 4-24 fold weaker in the asparagine enzyme. The turnover numbers were reduced by a factor of 70 for ethanol oxidation and 30 for acetaldehyde reduction at pH 7.3, 30/sup 0/C. Dead end inhibition studies and the kinetic isotope effect showed that NAD and ethanol binding follow a rapid equilibrium random mechanism as opposed to the ordered mechanism found for ADH I. They conclude that the carboxyl group of Asp-46 is essential for the electrostatic environment near the active site zinc. Amidation may affect the geometry and/or coordination of the metal complex.

• Research Organization: Univ. of Iowa, Iowa City
• OSTI ID: 5978039
• Report Number(s): CONF-870644-
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 46:6; Conference: 78. annual meeting of the American Society of Biological Chemists conference, Philadelphia, PA, USA, 7 Jun 1987
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ALCOHOL DEHYDROGENASE
ABSORPTION SPECTRA
BIOCHEMICAL REACTION KINETICS
ELECTROPHORESIS
ENZYME ACTIVITY
GENE MUTATIONS
MOLECULAR STRUCTURE
ASPARTIC ACID
AMINO ACID SEQUENCE
ASPARAGINE
BIOLOGICAL FUNCTIONS
FLUORESCENCE
LIGANDS
AMIDES
AMINO ACIDS
CARBOXYLIC ACIDS
ENZYMES
FUNCTIONS
HEMIACETAL DEHYDROGENASES
KINETICS
LUMINESCENCE
MUTATIONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXIDOREDUCTASES
REACTION KINETICS
SPECTRA
550200* - Biochemistry