Abstract
Data are presented, based upon subunit complementation experiments, that suggest that Escherichia coli adenylosuccinate synthetase contains two shared active sites between its dimeric interface. This conclusion was alluded to by use of mutant forms of adenylosuccinate synthetase previously prepared by site-directed mutagenesis. The experiments indicate that, although the R143L and D13A mutants have low or no activity independently, when they are mixed, a significant amount of activity was obtained. These results indicate that the subunits exchange with each other to form heterodimers with a single viable wild-type active site. The kcat value for the active hybrid active site in the R143L-D13A heterodimer is virtually identical to that observed with the wild-type enzyme, and the other kinetic parameters are very similar to those found for the wild-type enzyme. An analysis of the restoration of the activity in the presence of substrates suggests that GTP and IMP stabilize the dimeric structure of the protein. A comparison of the restoration of the activity using different combinations of mutants provides evidence indicating that some of the GTP binding elements, including the P-loop, in the protein are important for subunit integ...Continue Reading
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