Ref ID : 3947
Marilyn A. Niemann and John Berech, Jr.; Demonstration and Preliminary Characterization of an Enzyme Capable of the Further Metabolism of the Thymidine Catabolite, beta-Aminoisobutyric Acid, in Tetrahymena pyriformis Starin GL. J.Protozool. 28(4):447-453, 1981
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The enzyme that catalyzes the critical conversion of the proposed reductive thymidine catabolic end product, beta-aminoisobutyric acid, to the initial anabolic reutilization substrate, probably methylmalonic semialdehyde, was investigated to unambiguously substantiate the operation of a reductive pyrimidine catabolic and reutilization pathway in Tetrahymena pyriformis strain GL. Although most of the studies on the further metabolism of beta-aminoisobutyric acid in other organisms suggested transamination of beta-aminoisobutyric acid to methylmalonic semialdehyde followed by its further oxidation to methylmalonic acid, such a transaminating system could not be demonstrated. By means of a sensitive fluorometric assay system, however, a low, but significant amount of beta-aminoisobutyric acid oxidase activity was detected. This enzymatic activity exhibited the following characteristics in homogenates: good activity in alkaline 0.2M Tris-HCl buffer with a rather broad pH optimum ranging from 7.8 to 9.0; optimum activity at a temperature of 37 degrees C; stimulation on the addition of exogenous FAD; inhibition on the addition of divalent cations, EDTA, or PCMB; little stimulation on the addition of detergents; and no increase in activity on repeated freezing and thawing. In addition, crude preparations of this oxidase were found to be relatively stable when stored up to one week either refrigerated or frozen, to have a specific activity of 2.8 nmoles/min/mg of protein, and to have a Km of 3.6x10E-1 M for D,L-beta-aminoisobutyric acid. Whether this high Km is physiologically significant or not, however, will have to await further investigation. Preliminary (NH4)2SO4 fractionation and affinity chromatography studies also indicated that this enzyme appears to be a unique and specific oxidase whose activity is separable from other marker enzymes, including other oxidases.