Purification and feedback control of threonine deaminase activity of Rhodopseudomonas spheroides.

1. Threonine deaminase has been purified about 1400fold from extracts of the photosynthetic bacterium Rhodopseudomonas spheroides by ammonium sulfate fractionation and by chromatography on DEAE-cellulose and hydroxylapatite columns. The purified enzyme is almost completely resolved with respect to bound coenzyme, and the activity of holoenzyme is specific for pyridoxal phosphate. The enzyme is fairly stable in solution in the presence of Lisoleucine. 2. Kinetic studies indicate that there are more than one threonine-binding sites on the enzyme, and that substratesubstrate interactions may be influenced by feedback modifiers, e.g. isoleucine and valine, either by direct or indirect interactions. 3. The purified enzyme is subject to feedback inhibition by L-isoleucine and the inhibition due to L-isoleucine is reversed by low concentrations of L-valine. 4. In the absence of L-isoleucine, L-valine at low concentrations activates the deamination reaction, and the stimulatory effect is eliminated at high threonine concentration. At high L-valine concentrations, the enzyme activity is inhibited at all substrate concentrations tested; the substrate saturation curve is similar to that obtained when r.-isoleucine was the enzyme inhibitor. 5. Inhibition of enzyme activity by L-isoleucine or by high L-valine is counteracted by HgCL; Hg+f ions do not influence the stimulatory influence due to low levels of L-valine. Furthermore, the enzyme can be completely inactivated by incubation with HgCL, and the activity may be regenerated by addition of ,&mercaptoethanol. 6. It is concluded from these studies that there are two binding sites for valine per molecule of enzyme and that one of these binding sites is closely associated with a threoninebinding site, whereas, the second one is identical with or closely overlapping that of the isoleucine site.