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.
Here we present a study of the conformational changes of the quaternary structure of E. coli aspartate transcarbamoylase (ATCase), as monitored by time-resolved small-angle X-ray scattering (TR-SAXS), upon combining with substrates, substrate analogs, and nucleotide effectors at temperatures between 5 -22 °C, obviating the need for ethylene glycol. TR-SAXS time courses tracking the T → R structural change after mixing with substrates or substrate analogs appeared to be a single phase under some conditions and biphasic under other conditions, which we ascribe to multiple ligation states producing a time course composed of multiple rates. Increasing the concentration of substrates up to a certain point increased the T → R transition rate, with no further increase in rate beyond that point. Most strikingly after addition of PALA to the enzyme the transition rate was over one order of magnitude slower than with the natural substrates. These results on the homotropic mechanism are consistent with a concerted transition between structural and functional states of either lowaffinity low-activity or high-affinity high-activity for aspartate. Addition of ATP along with the substrates increased the rate of the transition from the T to the R state and also decreased the duration of the R-state steady-state phase. Addition of CTP or the combination of CTP/UTP to the substrates significantly decreased the rate of the T → R transition and caused a shift in the enzyme population towards the T state even at saturating substrate concentrations. These results on the heterotropic mechanism suggest a destabilization of the T state by ATP and a destabilization of the R state by CTP and CTP/UTP, consistent with the T and R state crystallographic structures of ATCase in the presence of the heterotropic effectors.
X-ray crystallography and small-angle X-ray scattering (SAXS) in solution have been used to show that a mutant aspartate transcarbamoylase exists in an intermediate quaternary structure between the canonical T and R structures. Additionally, the SAXS data indicate a pH-dependent structural alteration consistent with either a pH-induced conformational change or a pH-induced alteration in the T to R equilibrium. These data indicate that this mutant is not a model for the R state, as has been proposed, but rather represents the enzyme trapped along the path of the allosteric transition between the T and R states.A lthough many aspects of allosteric regulation and cooperativity have been established for Escherichia coli aspartate transcarbamoylase (ATCase), the enzyme that catalyzes the committed step in pyrimidine nucleotide biosynthesis, many of the details of how the active sites change from low-activity, lowaffinity to high-activity, high-affinity during the T to R transition have not been delineated (1). The major limitation has been that stabilization of the enzyme in the R state requires the presence of active site ligands, thus making interpretation of the structural rearrangements upon the T to R transition, in the absence of ligands, difficult. Newell and Schachman (2) have concluded, from sedimentation velocity experiments, that the quaternary structures of ATCase with the catalytic chain mutation K164E and the catalytic chain double-mutation K164E and E239K exist in the R state, irrespective of the presence of active site ligands. The enzymatic properties of the double mutant (K164E/E239K ATCase) include a lack of homotropic cooperativity and an inability to be activated by ATP or inhibited by CTP: properties coincident of an enzyme that cannot transition between the two allosteric states (2). More recently, Velyvis et al. (3) demonstrated that the partially labeled K164E/E239K ATCase exhibited certain chemical shifts in solution NMR studies that they conclude are characteristic of the R state of the enzyme. These same chemical shifts were also observed when the wild-type enzyme was bound with the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) (3), which is known to stabilize fully the R state of the enzyme (4). Thus, a crystal structure of K164E/ E239K ATCase should provide us a means to obtain the structural details of an R-state active site in the absence of ligands. However, here we report the X-ray crystal structure and the solution small-angle X-ray scattering (SAXS) data for the K164E/ E239K ATCase, which clearly demonstrate that this doublemutant enzyme is not in the R-quaternary structure. Instead this mutant enzyme is in a unique intermediate state on the path between the T and R structural states. Results and DiscussionCrystal Structure of K164E/E239K ATCase Is Different from the WildType R State Structure. The K164E/E239K ATCase was purified and subsequently crystallized in the absence of ligands as described in Methods. The crystals were determined to be in the P2 1 2 1 2 1 space gro...
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