Identity and some properties of the L-threonine aldolase activity manifested by pure 2-amino-3-ketobutyrate ligase of Escherichia coli.
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 degrees C in the absence, as well as in the presence of acetyl CoA, a protective substrate; (c), ratios of the two enzymatic 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 Km values for glycine (ligase) and L-threonine (aldolase) are 10 mM and 0.9 mM, respectively, whereas corresponding respective Vmax values were found to be 2.5 mumol of CoA released/min per mg vs. 0.014 mumol of acetaldehyde formed (NADH oxidized)/min per mg.
Marcus
J P
JP
Department of Biological Chemistry, University of Michigan, Ann Arbor.
Dekker
E E
EE
D000123:Acetyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology*; D005998:Glycine / Q000737:chemistry; D012696:Glycine Hydroxymethyltransferase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D011732:Pyridoxal Phosphate; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry
D013912:Threonine; D011732:Pyridoxal Phosphate; D012696:Glycine Hydroxymethyltransferase; D000123:Acetyltransferases; C020679:glycine acetyltransferase; D005998:Glycine; D007460:Iodoacetamide