Open AccessAlanine dehydrogenase from Streptomyces fradiae
Author(s) -
VANČURA Aleš,
VANČUROVÁ Ivana,
VOLC Jindřich,
JONES Shanagh K. T.,
FLIEGER Miroslav,
BASAŘOVÁ Gabriela,
BĚHAL Vladislav
Publication year - 1989
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1989.tb14544.x
Subject(s) - isoelectric point , chemistry , oxidative deamination , alanine , chromatography , dehydrogenase , nad+ kinase , lactate dehydrogenase , biochemistry , enzyme , amino acid
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 210000 or 205000 by equilibrium ultracentrifugation or gel filtration, respectively. The enzyme is composed of four subunits, each of M r 51000. Using analytical isoelectric focusing the isoelectric point of alanine dehydrogenase was found to be 6.1. The K m were 10.0 mM for L ‐alanine and 0.18 mM for NAD + . K m values for reductive amination were 0.23 mM for pyruvate, 11.6 mM for NH + 4 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.