L. Malkin, D. M. Greenberg
Apr 6, 1964
Citations
1
Influential Citations
48
Citations
Quality indicators
Journal
Biochimica et biophysica acta
Abstract
1. 1. Allothreonine (l-allothreonine acetaldehyde-lyase, EC 4.1.2.6) or threonine aldolase (l-threonine acetaldehyde-lyase, EC4.1.2.5), an enzyme that cleaves both allothreonine and threonine to glycine and acetaldehyde, has been purified 174-fold from rat liver using acetone precipitation, (NH4)2SO4 fractionation and negative adsorption with hydroxyapatite gel. The purified preparation was found to be free of threonine deaminase (l-threonine hydro-lyase (deaminating), EC 4.2.1.16). 2. 2. That allothreonine and threonine are degraded by the same enzyme was demonstrated by the ratios of allothreonine/threonine activities remaining constant during purification, inactivation on standing in buffers of various Ph, and during removal and readdition of the cofactor, pyridoxal phosphate. In addition, the rates were not additive when both substrates were assayed together. 3. 3. The Km for l-threonine was calculated to be 20 mM, for l-allothreonine, 0.217 mM. In addition, the reaction rate with allothreonine was 1.8- to 2.0 fold greater than the rate with threonine when both were at saturating levels. 4. 4. Pyridoxal phosphate was shown to be cofactor for the enzyme by demonstrating a decrease in activity after treatment with hydroxylamine or charcoal and a partial reactivation upon the addition of the cofactor. Pyridoxal phosphate also appears to have a general protective effect as it protects the enzyme against inactivation by dilution and prevents enzyme solutions from turning turbid on standing in phosphate buffer. 5. 5. Allothreonine was found to be a small contaminant of commercial threonine but it did not account for all of the activity observed with threonine as a substrate. 6. 6. Reversibility of the reaction was demonstrated with allothreonine only; the absence of formation of threonine in the reverse reaction was determined by a microbiological assay procedure using Streptococcus faecalis R. 7. 7. Aldolase activity towards β-phenylserine is present in the purified preparation, but varying β-phenylserine/allothreonine ratios during purification and the fact that the rates were additive when both substrates were assayed together demonstrated that β-phenylserine aldolase is a different enzyme than allothreonine adolase.