Biodegradative threonine dehydratase of Salmonella typhimurium LT2 has been purified by affinity chromatography on AMP-Sepharose. In the presence of AMP, an allosteric effector, the enzyme exhibits a molecular weight of about 140 000 and is composed of four subunits of 36 000 each. The purified protein showed an absorbance maximum at 413 nm characteristic of enzymes containing bound pyridoxal phosphate; chemical determination indicated 2 mol of pyridoxal phosphate per 140 000 g. The Km value for pyridoxal phosphate for the apoenzyme was 5 μM. Titrations of sulfhydryl groups with and without sodium dodecyl sulfate showed three-SH groups per 36 000 g, one of which is “buried”. The catalytic activity of the enzyme was stimulated by AMP as well as by CMP, GMP, UMP, and ADP. The Km for l-threonine was decreased by 15-fold when assayed with 3 mM AMP relative to that without AMP; the ratio of Vmax values with and without AMP was 6. The enzyme activity was reversibly inhibited by pyruvate in the pre ence of AMP and revealed a strictly noncompetitive inhibition with respect to l-threonine and a “mixed” kinetic relationship in terms of AMP. Inhibition by pyruvate was independent of enzyme concentration. Incubation of the enzyme under assay conditions with [14C] pyruvate for 5 min showed no protein-bound radioactivity. In addition to allosteric regulation, the threonine dehydratase was also subjected to catabolite inactivation by several intermediary metabolites, including pyruvate. The rate of enzyme inactivation was decreased with increasing protein concentration; AMP was obligatorily required for the inactivation process. Gel-filtration data showed that enzyme inactivation by pyruvate led to dissociation of the native enzyme; the inactive dissociated form of the enzyme contained 1 mol of firmly bound pyruvate per 140 000 g. Dialysis of the dissociated form of the enzyme against AMP and dithiothreitol resulted in reassociation to yield active enzyme with pyruvate still attached to the protein. We conclude that, despite the overall similarity in the control of biodegradative threonine dehydratases from Escherichia coli and S. typhimurium, the molecular mechanisms for the allosteric inhibition and catabolite inactivation by pyruvate of the enzymes from these closely related organisms are distinct and presumably reflect the differences in the structural features and the association-dissociation behavior of these two proteins. © 1978, American Chemical Society. All rights reserved.
