Application of methyl-TROSY NMR to test allosteric models describing effects of nucleotide binding to aspartate transcarbamoylase.

Aspartate transcarbamoylase has emerged as a textbook example of an allosteric enzyme whose binding of active-site substrates can be explained on the basis of the classical Monod-Wyman-Changeux (MWC) model of allostery. There is still debate, however, regarding the mode of action of ATP and cytidine triphosphate (CTP)--allosteric effectors that bind at regulatory sites 60 A away from the nearest active site. A large body of data for nucleotide binding is consistent with the MWC model, including a previous NMR study showing a shift in the allosteric equilibrium between R and T states that is predicted by this scheme. The possibility of binding-promoted changes to the structures of the active sites, while not within the framework of the MWC model, cannot be excluded, however. Here, the effects of binding of nucleotides are monitored in a series of (1)H-(13)C methyl transverse relaxation optimized spectroscopy spectra recorded on the 300-kDa aspartate transcarbamoylase holoenzyme in both the absence and the presence of saturating amounts of ATP or CTP. No changes in shifts of methyl probes of the catalytic chains (c-chains) that include the active sites are observed, consistent with a lack of structural changes. In addition, methyl (1)H-(13)C residual dipolar couplings are measured that are exquisitely sensitive to methyl axis orientations, and correlations between couplings measured on samples with and without nucleotide show no changes in structure of the c-chains. These results indicate that the mechanism of action of ATP and CTP can be explained fully by the MWC model and that any scheme invoking structural changes of the c-chains is not correct.