The functional unit of polyenzymes. Determination by radiation inactivation

Abstract

Recently, target analysis has been re-evaluated as a technique for the determination of molecular sizes (Kempner, E. S. & Schlegel, W. (1979) Anal. Biochem. 92, 2-10). The technique yields the size of the functional unit, i.e. the minimal assembly of structures necessary for a given function such as an enzymatic activity. Using this method, we have not determined the sizes of the functional units for different enzymatic activities on the "arom" conjugate from Euglena, a polyenzyme catalyzing five sequential reactions in the shikimic acid pathway, and on two conjugates from Escherichia coli carrying both aspartokinase and homoserine dehydrogenase activities. In each conjugate, the size for different enzymatic activities was measured and found to be the same. When compared to the molecular weight obtained with other techniques, the target size matched either the entire conjugate (aspartokinase-homoserine dehydrogenase conjugates I and II) or half the unit ("arom" conjugate). The information was obtained with minimal perturbation of the complexes and sparing laborious purification and reconstitution experiments. Tryptophan synthase was irradiated both as an intact conjugate and also as isolated subunits. In both structural forms, beta 2 was identified as the functional unit for the conversion of indole and serine to tryptophan. The results of this study give insight into the structural assembly of these polyenzymes.