Identity and some properties of the l-threonine aldolase activity manifested by pure 2-amino-3-ketobutyrate ligase of Escherichia coli

Abstract

2-Amino-3-ketobutyrate ligase catalyzes the reversible, pyridoxal 5′-phosphate-dependent condensation of glycine with acetyl CoA forming the unstable intermediate, 2-amino-3-ketobutyrate. Several independent lines of evidence indicate that the pure protein obtained in the purification of this ligase from Escherichia coli also has l-threonine aldolase activity. The evidence includes: (a), a constant ratio of specific activities (aldolase/ligase) at all stages of purifying 2-amino-3-ketobutyrate ligase to homogeneity; (b), the same rate of loss of aldolase and ligase activities during controlled heat inactivation of the pure protein at 60°C in the absence, as well as in the presence of acetyl CoA, a protective substrate; (c), ratios of the two enzymic activities that are not significantly different during slow inactivation by iodoacetamide, with and without l-threonine added; (d), coincident rates of loss and essentially identical rates of recovery of aldolase activity and ligase activity during resolution of the holoenzyme with hydroxylamine followed by reconstitution with pyridoxal 5′-phosphate. No aldolase activity is observed with d-threonine as substrate and l-allothreonine is about 25% as effective as l-threonine. Whereas ligase activity has a sharp pH optimum at 7.5, the aldolase activity of this pure protein is maximal at pH 9.0. Comparative apparent K m values for glycine (ligase) and l-threonine (aldolase) are 10 mM and 0.9 mM, respectively, whereas corresponding respective V max values were found to be 2.5 μmol of CoA released/min per mg vs. 0.014 μmol of acetaldehyde formed (NADH oxidized)/min per mg.