title: Alanine dehydrogenase from Streptomyces fradiae. Purification and properties.
abstract: Alanine dehydrogenase was purified to homogeneity from a cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1180-fold to give a 21% yield, using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The relative molecular mass of the native enzyme was determined to be 210,000 or 205,000 by equilibrium ultracentrifugation or gel filtration, respectively. The enzyme is composed of four subunits, each of Mr 51,000. Using analytical isoelectric focusing the isoelectric point of alanine dehydrogenase was found to be 6.1. The Km were 10.0 mM for L-alanine and 0.18 mM for NAD+. Km values for reductive amination were 0.23 mM for pyruvate, 11.6 mM for NH4+ and 0.05 mM for NADH. Oxidative deamination of L-alanine proceeds through a sequential-ordered binary-ternary mechanism in which NAD+ binds first to the enzyme, followed by alanine, and products are released in the order ammonia, pyruvate and NADH.
authors.affiliation: Prague Institute of Chemical Technology, Czechoslovak Academy of Sciences.
authors.forename: A
authors.initials: A
authors.lastname: Vancura
authors.forename: I
authors.initials: I
authors.lastname: Vancurová
authors.forename: J
authors.initials: J
authors.lastname: Volc
authors.forename: S K
authors.initials: SK
authors.lastname: Jones
authors.forename: M
authors.initials: M
authors.lastname: Flieger
authors.forename: G
authors.initials: G
authors.lastname: Basarová
authors.forename: V
authors.initials: V
authors.lastname: Bĕhal
mesh_terms: D050788:Alanine Dehydrogenase; D000586:Amination; D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D014461:Ultracentrifugation
chemical_list: D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase