L-Threonine dehydrogenase of chicken liver. Purification, characterization, and physiological significance.

Abstract

L-Threonine dehydrogenase (L-threonine:NAD' oxidoreductase (EC 1.1.1.103))has been purified to apparent homogeneity from chicken liver mitochondria.The presence of 2-mercaptoethanol and glycerol is necessary for stabilizing the enzyme during purification and storage.The enzyme is a monomer and has M, 88,000.The pH optimum is 8.6 to 8.7, and the isoelectric point of the enzyme is 5.9.The enzyme is specific for L-threonine and NAD+.The K,,, values for L-threonine and NAD' are 8.4 and 0.98 m, respectively.Kinetic studies indicate that the reaction proceeds through an Ordered Bi Bi mechanism where NAD+ is added first, followed by L-threonine.Chicken liver mitochondria contain aminoacetone synthase (acetyl-CoA:glycine C-acetyltransferase (EC 2.3.1.29)).The enzyme has been partially purified.In the presence of L-threonine dehydrogenase and aminoacetone synthase, L-threonine is cleaved to glycine and acetyl-coA via 2-amino-3-oxobutyrate.The result suggests that L-threonine dehydrogenase and aminoacetone synthase have a physiological role in L-threonine metabolism in vertebrates.L-Threonine dehydrogenase catalyzes NAD'-dependent oxidation of L-threonine.The suspected product of the reaction is L-2-amino-3-oxobutyrate which spontaneously decomposes to yield aminoacetone and COz (1).The dehydrogenase appears to function as the first step in a threonine degradation pathway in several microorganisms that use L-threonine as the main carbon and energy source (2-7).In microorganisms, 2-amino-3-oxobutyrate is further cleaved in a CoA-dependent reaction to produce glycine and acetyl-coA (3).L-Threonine dehydrogenase has been purified to homogeneity from Escherichia coli K-12 (8).The enzyme has M , = 140,000 and consists of four identical subunits.To date, the enzyme has been only partially purified from animal sources.The enzyme from bullfrog liver has been purified to 7.4-fold with a specific activity of 339 nmol of aminoacetone formed/ min/mg of protein (9).Recent studies (10, 11) suggested that guinea pig and rat can degrade L-threonine to glycine and acetyl-coA by the pathway found in microorganisms.The role and the mechanism of L-threonine dehydrogenase reaction in animals, however, have not been fully elucidated by the lack of highly purified enzyme from animals.In this communication, we describe the purification and properties of homogeneous L-threonine dehydrogenase from