Conformational Changes in Aspartate Transcarbamylase: II. Concerted or sequential mechanism?

Abstract

Abstract Tryptic digestion rates of aspartate transcarbamylase, as well as reaction rates of p-mercuribenzoate with the thiol groups of this enzyme, were determined as a function of aspartate or succinate concentration. The two methods gave identical for a given ligand, indicating that increased digestibility and increased availability of thiol groups both reflect the same ligand-induced conformational transition. The shape of the state function given by aspartate alone was markedly different from that obtained with succinate in the presence of carbamyl phosphate. State functions for aspartate determined by digestibility were compared with saturation functions for aspartate obtained by activity measurements in the presence of carbamyl phosphate. This was done by plotting against each other the substrate concentrations required to reach the same degree of completion of the two functions. The resulting plot was linear throughout its entire course indicating that the shapes of the state and the corresponding saturation functions were identical, i.e. the curves were homologous. Such homology is not compatible with a concerted mechanism for allosteric transitions, but is consistent with a sequential mechanism. In fact, calculations using a wide range of assumptions for the concerted mechanism showed that this model cannot generate homologous state and saturation functions, and therefore cannot account for the behavior observed when aspartate was used as the ligand. In contrast to the apparent sequential behavior with aspartate, the data obtained by Gerhart and Schachman (Biochemistry, 7, 538 (1968)) using succinate (in the presence of carbamyl phosphate) were compatible with a concerted mechanism. It seems therefore that whether aspartate transcarbamylase will follow one, or the other, of these mechanisms depends on the ligand which occupies the catalytic site. The conformational effect of the allosteric activator, ATP was also investigated, and is discussed in relation to allosteric models.