A study of threonine deaminase from Saccharomyces cerevisiae has been undertaken. The enzyme has been purified 50-fold, with a low resolution of pyridoxal phosphate coenzyme. The purified enzyme is very unstable under all conditions tried so far. Properties have been studied with crude extracts. Optimum pH has been found to be 8.2. Isoleucine has complex effects on the enzyme activity. At a concentration of 0.1 mM, it is able to stimulate the enzyme. At higher concentrations, isoleucine inhibits. The Ki values increase with increasing pH, but 100% inhibition can be reached at all ranges of pH studied. Affinity of the enzyme for its substrate is even more strongly modified by pH. a) Substrate cooperativity, nil at pH 7, intermediate at pH 8, seems to be maximum at pH 9. At any pH, the substrate cooperativity can be suppressed by addition of L-valine. In addition, rather low concentrations of isoleucine also act partly as valine does. b) Apparent Km values, measured in the presence of valine, vary with pH in the opposite direction to Ki values. Hill's coefficients for substrate and inhibitor reflect these findings. For threonine alone, they vary from 1.2 at pH 7 to 3 at pH 9, In the presence of valine, they remain close to 1, at all pH. Isoleucine, 0.1 mM, produces an incomplete effect compared to valine. For isoleucine inhibition, the cooperativity coefficients remain close to 3 at all pH. The molecular weight of the enzyme has been determined to be roughly 1.2 ? 105, at, pH 6.8 and at pH 8.7. Thermal inactivation is unimodal and fails to show desensitization of the enzyme in any of the conditions tried. The implications of these results on the structure of the enzyme have been discussed.

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Journal Article

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Robichon-Szulmajster H, Magee PT

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