Biosynthesis of branched-chain amino acids in Schizosaccharomyces pombe: regulatory properties of threonine deaminase.
Biosynthetic threonine deaminase (TD) from Schizosaccharomyces pombe has been partially purified from crude extracts by treatment with protamine sulfate, ammonium sulfate precipitation, and gel filtration through Sephadex G-25. In both crude extracts and purified preparations, TD showed marked stimulation by pyridoxal phosphate. A pH optimum for activity was found at pH 9.0, whereas the inhibition caused by the natural feedback inhibitor, l-isoleucine, was maximal at pH 7.4. l-Threonine exhibits homotropic cooperative effects at low pH (7.0-8.0), which are eliminated at pH 9.0, and the affinity for substrate (in terms of K(m)) increased with increasing pH. Enzyme activity could be completely inhibited by isoleucine over a pH range of 7.4 to 9.0; the amount of isoleucine required for 50% inhibition increased with increasing pH. Isoleucine inhibition was pseudocompetitive with respect to substrate and increased the cooperative effects of threonine. l-Valine was found to reverse isoleucine inhibition; it also activated the enzyme in a pH range of 7.0 to 8.0 by eliminating the cooperative effects of threonine, thus normalizing the substrate saturation curves at these pH values. l-Leucine was shown to be a competitive inhibitor with respect to threonine, and to be able partially to reverse isoleucine inhibition. Treatment of TD with mercurials did not result in desensitization to isoleucine inhibition. However, at pH 10, virtually no sensitivity of the enzyme to isoleucine was observed while activity remained strong, which suggests the existence of separate sites on the TD molecule for binding threonine and isoleucine. A tentative model is presented which unifies the kinetic results reported here in terms of the interactions of TD with its effector molecules.
D000409:Alanine / Q000494:pharmacology; D000596:Amino Acids / Q000096:biosynthesis*; D000645:Ammonium Sulfate; D001203:Ascomycota / Q000201:enzymology*; D002850:Chromatography, Gel; D003470:Culture Media; D004789:Enzyme Activation; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007532:Isoleucine / Q000494:pharmacology; D007930:Leucine / Q000494:pharmacology; D011479:Protamines; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D013268:Stimulation, Chemical; D013696:Temperature; D013912:Threonine; D014633:Valine / Q000494:pharmacology
D000596:Amino Acids; D003470:Culture Media; D011479:Protamines; D007532:Isoleucine; D013912:Threonine; D011732:Pyridoxal Phosphate; D006836:Hydro-Lyases; D007930:Leucine; D014633:Valine; D000409:Alanine; D000645:Ammonium Sulfate