THREONINE FORMATION VIA THE COUPLED ACTIVITY OF 2-AMINO-3-KETOBUTYRATE COENZYME-A LYASE AND THREONINE DEHYDROGENASE

The enzymes L-threonine dehydrogenase and 2-amino-3-ketobutyrate coenz yme A (CoA) lyase are known to catalyze the net conversion of L-threon ine plus NAD+ plus CoA to NADH plus glycine plus acetyl-CoA. When homo geneous preparations of these two enzymes from Escherichia coli were i ncubated together for 40 min at 25-degrees-C with glycine, acetyl-CoA, and NADH, a 36% decrease in the level of glycine (with concomitant NA DH oxidation) was matched by formation of an equivalent amount of thre onine, indicating that this coupled sequence of enzyme-catalyzed react ions is reversible in vitro. Several experimental factors that affect the efficiency of this conversion in vitro were examined. A constructe d strain of E. coli, MD901 (glyA thrB/C tdh), was unable to grow unles s both glycine and threonine were added to defined rich medium. Introd uction of the plasmid pDR121 (tdh+ kbl+) into this strain enabled the cells to grow in the presence of either added glycine or threonine, in dicating that interconversion of these two amino acids occurred. Threo nine that was isolated from the total pool of cellular protein of MD90 1/pDR121 had the same specific radioactivity as the [C-14] glycine add ed to the medium, establishing that threonine was formed exclusively f rom glycine in this strain. Comparative growth rate studies with sever al strains of E. coli containing plasmid pDH121, together with the fin ding that k(cat) values of pure E. coli 2-amino-3-ketobutyrate CoA lya se favor the cleavage of 2-amino-2-ketobutyrate over its formation by a factor of 50, indicate that the biosynthesis of threonine is less ef ficient than glycine formation via the coupled threonine dehydrogenase -2-amino-3-ketobutyrate lyase reactions.