New Paradigm for Allosteric Regulation of Escherichia coli Aspartate Transcarbamoylase

Abstract

For nearly 60 years, the ATP activation and the CTP inhibition of Escherichia coli aspartate transcarbamoylase (ATCase) has been the textbook example of allosteric regulation. We present kinetic data and five X-ray structures determined in the absence and presence of a Mg(2+) concentration within the physiological range. In the presence of 2 mM divalent cations (Mg(2+), Ca(2+), Zn(2+)), CTP does not significantly inhibit the enzyme, while the allosteric activation by ATP is enhanced. The data suggest that the actual allosteric inhibitor of ATCase in vivo is the combination of CTP, UTP, and a divalent cation, and the actual allosteric activator is a divalent cation with ATP or ATP and GTP. The structural data reveals that two NTPs can bind to each allosteric site with a divalent cation acting as a bridge between the triphosphates. Thus, the regulation of ATCase is far more complex than previously believed and calls many previous studies into question. The X-ray structures reveal that the catalytic chains undergo essentially no alternations; however, several regions of the regulatory chains undergo significant structural changes. Most significant is that the N-terminal region of the regulatory chains exists in different conformations in the allosterically activated and inhibited forms of the enzyme. Here, a new model of allosteric regulation is proposed.

Strain analysis of protein structures and low dimensionality of mechanical allosteric couplings

“…4A). The enzymatic activity of ATCase is allosterically regulated by the binding of one or two nucleotides to each regulatory subunit, with purines (adenosine and guanosine bases) enhancing activity and pyrimidines (cytidine and thymidine bases) inhibiting activity (49).…”

X-ray Scattering Studies of Protein Structural Dynamics

“…A later study by Cockrell et al demonstrated the importance of Mg 2+ as a cation for the nucleotide effectors. 147 In this work, it was shown that although CTP on its own can inhibit the enzyme, activity is restored with addition of physiological levels of Mg 2+ . Instead, the highest degree of inhibition was observed with the combination of the pathway products CTP and UTP in the presence of Mg 2+ (Figure 10).…”

“…As noted by Cockrell et al , the predicted scattering of the PALA-bound structure with Mg 2+ -ATP (Figure 14, black dotted) is slightly greater than that of the structure with only PALA bound (Figure 14, black solid). 147 However, neither of the predicted curves describes the experimentally obtained scattering of PALA-bound ATCase in solution with (Figure 14, red) or without (Figure 14, orange) Mg 2+ -ATP.…”

“…136 However, the scattering of ATCase with PALA (orange curve) or with PALA and Mg 2+ -ATP (red curve) do not overlay with theoretical profiles of crystal structures with PALA-bound (black solid, PDB: 1D09) 137 or with PALA and Mg 2+ -ATP bound (black dotted, PDB: 4KH0). 147 As noted by Svergun, 138 this discrepancy is likely due to an R-state more open in solution than in a crystal. Adapted with permission from Ref.…”

“…139 Thus, a purely crystallographic argument may not take into account the added flexibility observable only via solution scattering. With the crystal structures from Cockrell et al 147 and SAXS profiles from Fetler et al 148 now available, this question can be revisited. Again, we find that the experimental scattering of the unliganded T-state (Figure 14, green) can be fit well with the predicted scattering of the crystal structure of ligand-free ATCase (Figure 14, gray).…”