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The catalytic subunit prepared by treatment of native aspartate transcarbamylase with />-mercuribenzoate has been used to investigate the mechanism of the reaction. The results of initial velocity, product inhibition, dead-end inhibition, isotope transfer, and binding studies are consistent with the reaction having a random mechanism for which the rate of interconversion of the central complexes appears to be slow compared with all other steps of the reaction sequence. Further, they indicate that the mechanism involves the formation of three dead-end complexes: enzyme-aspartate-Pi, enzyme-aspartate-carbamyl aspartate, and enzyme-carbamyl phosphate-carbamyl aspartate. In addition, initial velocity and product inhibition studies have been made using the i3oth the native form of aspartate transcarbamylase, isolated from Escherichia coli, and the catalytic subunit derived from it by treatment with /j-mercuribenzoate catalyze the reaction: carbamyl phosphate + aspartatecarbamyl aspartate + Pi. From investigations of the binding of succinate, an inhibitory analog of aspartate, to the catalytic subunit, it was reported that the binding was dependent on the presence of carbamyl phosphate (Changeux et al., 1968). Thus, it was inferred that the reaction involving aspartate would occur via an ordered mechanism with carbamyl phosphate as the first substrate to add to the enzyme. This conclusion was supported by the results of steady-state kinetic studies from which it was concluded that carbamyl aspartate was released from the enzyme before P¡ (Porter et al., 1969).By contrast with the above findings, the results of Collins and Stark (1969) showed that succinate did combine with the catalytic subunit although its reaction was considerably enhanced in the presence of carbamyl phosphate. In addition, these authors showed that the catalytic subunit is capable of binding aspartate. Moreover, aspartate influences the rate of digestion of the native enzyme by proteolytic enzymes (Mc-Clintock and Markus, 1968,1969). These results raise the possibility that substrates bind in a random manner to both the native enzyme and catalytic subunit.At the time of publication of the paper by Porter et al. (1969), steady-state kinetic investigations were being carried out with the catalytic subunit which can be obtained by limited t From the

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