Structure and function of aspartate transcarbamoylase studied using chymotrypsin as a probe

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Abstract
Aspartate transcarbamoylase [EC 2.1.3.2] from Escherichia coli is composed of 6 catalytic (c) and 6 regulatory (r) polypeptides. The structure and function of this enzyme were studied using chymotrypsin as a probe. The protease inactivates the isolated catalytic subunit (c3) but has no effects on the native enzyme (c6r6). Under identical conditions, the c3r6 complex is inactivated at a much slower rate than c3. The presence of the substrate analog succinate together with carbamoyl phosphate reduces substantially the rate of inactivaton. Extended exposure to chymotrypsin converts the catalytic subunit into a partially active derivative with a 4-fold higher Km. This derivative is indistinguishable from the unmodified catalytic subunit in gel electrophoresis under nondenaturing conditions. In the presence of sodium dodecyl sulfate, the major fragment in the electropherogram is smaller than that of the intact catalytic polypeptide. The results could be explained by postulating the presence of a chymotrypsin-sensitive peptide bond at or near the active site. Since X-ray crystallographic studies indicated that the active sites are located in a central cavity, the resistance of the native enzyme towards inactivation may be due to the inability of chymotrypsin to enter this cavity.

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