Here we use the fluorescence from a genetically encoded unnatural amino acid, L-(7-hydroxycoumarin-4-yl)ethylglycine (HCE-Gly) replacing an amino acid in the regulatory site of Escherichia coli aspartate transcarbamoylase (ATCase) to decipher the molecular details of regulation of this allosteric enzyme. The fluorescence of HCE-Gly is exquisitely sensitive to the binding of all four nucleotide effectors. Although ATP and CTP are primarily responsible for influencing enzyme activity, the results of our fluorescent binding studies indicate that UTP and GTP bind with similar affinities, suggesting a dissociation between nucleotide binding and control of enzyme activity. Furthermore, while CTP is the strongest regulator of enzyme activity, it binds selectively to only a fraction of regulatory sites, allowing UTP to effectively fill the residual ones. Our results suggest that CTP and UTP are not competing for the same binding sites, but instead reveal an asymmetry between the two allosteric sites on the regulatory subunit of the enzyme. Correlation of binding and activity measurements explain how ATCase uses asymmetric allosteric sites to achieve regulatory sensitivity over a broad range of heterotropic effector concentrations.Enzymes responsible for catalyzing the committed step of critical metabolic pathways, such as aspartate transcarbamoylse (ATCase) in pyrimidine nucleotide biosynthesis, are often regulated by allosteric mechanisms. Allosteric regulation provides a rapid means for turning a pathway on or off dictated by the specific needs of the cell at any point in time. Escherichia coli ATCase has been investigated extensively and has come to serve as a paradigm for allosteric enzymes (1). ATCase is regulated by both homotropic and heterotropic effectors involving the cooperative binding of the second substrate, aspartate, at the active site and nucleotide binding at the allosteric site. The active and allosteric sites are located ~60Å apart on different polypeptide chains of the holoenzyme, a dodecamer composed of six catalytic chains (c) and six regulatory chains (r) (Figure 1, panel a), which can be dissociated into two catalytically active trimers (c 3 ) and three nucleotide-binding regulatory dimers (r 2 ) (2).Kinetic studies revealed activation by ATP, inhibition by CTP, and the synergistic inhibition of UTP in the presence of CTP (3). However, the elucidation of the allosteric mechanism of regulation in ATCase has been exceedingly difficult with many conflicting results. The interactions of the regulatory nucleotides with the enzyme have been investigated using a variety of methods including equilibrium dialysis (4-8), continuous-flow dialysis (9), nuclear magnetic resonance (10,11), site-specific mutagenesis (8,10-13), and X-ray crystallography (14,15). Some of these experiments indicate two classes of nucleotide binding sites with differing affinities suggesting either a non-equivalence of binding sites or negative cooperativity (7). However, these methods lack the sensitivity to accurately * ...

Here we report the isolation, kinetic characterization, and X-ray structure determination of a cooperative E. coli aspartate transcarbamoylase (ATCase) without regulatory subunits. The native ATCase holoenzyme consists of six catalytic chains organized as two trimers bridged noncovalently by six regulatory chains organized as three dimers, c 6 r 6 . Dissociation of the native holoenzyme produces catalytically active trimers, c 3 , and nucleotide-binding regulatory dimers, r 2 . By introducing specific disulfide bonds linking the catalytic chains from the upper trimer site specifically to their corresponding chains in the lower trimer prior to dissociation, a new catalytic unit, c 6 , was isolated consisting of two catalytic trimers linked by disulfide bonds. Not only does the c 6 species display enhanced enzymatic activity compared to the wild-type enzyme, but the disulfide bonds also impart homotropic cooperativity, never observed in the wild-type c 3 . The c 6 ATCase was crystallized in the presence of phosphate and its X-ray structure determined to 2.10 Å resolution. The structure of c 6 ATCase liganded with phosphate exists in a nearly identical conformation as other R-state structures with similar values calculated for the vertical separation and planar angles. The disulfide bonds linking upper and lower catalytic trimers predispose the active site into a more active conformation by locking the 240's loop into the position characteristic of the high-affinity R state. Furthermore, the elimination of the structural constraints imposed by the regulatory subunits within the holoenzyme provides increased flexibility to the c 6 enzyme enhancing its activity over the wild-type holoenzyme (c 6 r 6 ) and c 3 . The covalent linkage between upper and lower catalytic trimers restores homotropic cooperativity so that a binding event at one or so active sites stimulates binding at the other sites. Reduction of the disulfide bonds in the c 6 ATCase results in c 3 catalytic subunits that display similar kinetic parameters to wild-type c 3 . This is the first report of an active c 6 catalytic unit that displays enhanced activity and homotropic cooperativity.Escherichia coli aspartate transcarbamoylase (ATCase, EC 2.1.3.2) catalyzes the committed step of the pyrimidine nucleotide biosynthesis pathway: the condensation of carbamoyl phosphate (CP) and L-aspartate (Asp) to form N-carbamoyl-L-aspartate (CA) and phosphate (P i ). The end products of the pyrimidine biosynthetic pathway, CTP and UTP in the presence of CTP, allosterically inhibit the enzyme (1,2). Conversely ATP, an end product of the purine biosynthetic pathway, allosterically activates the enzyme (1).The quaternary structure of the E. coli enzyme is a dodecamer composed of two trimeric catalytic subunits (M r = 34,000/chain) and three dimeric regulatory subunits (M r = 17,000/ † This work was supported by National Institutes of Health (GM26237). Use of the National Synchrotron Light Source, Brookhaven National Laboratory, was supported by the U.S. (20). T...

The pathway of product release from the R state of aspartate transcarbamoylase has been determined here by solving the crystal structure of Escherichia coli aspartate transcarbamoylase (ATCase) locked in the R-quaternary structure by specific introduction of disulfide bonds. ATCase displays ordered substrate binding and product release, remaining in the R state until substrates are exhausted. The structure reported here represents ATCase in the R state bound to the final product molecule, phosphate. This structure has been difficult to obtain previously because the enzyme relaxes back to the T state after the substrates are exhausted. Hence cocrystallizing the wild-type enzyme with phosphate results in a T-state structure. In this structure of the enzyme trapped in the R state with specific disulfide bonds, we observe two phosphate molecules per active site. The position of the first phosphate corresponds to the position of the phosphate of carbamoyl phosphate and the position of the phosphonate of N-phosphonacetyl-L-aspartate. However, the second, more weakly bound phosphate, is bound in a positively charged pocket more accessible to the surface than the other phosphate. The second phosphate appears to be on the path that phosphate would have to take to exit the active site. Our results suggest that phosphate dissociation and carbamoyl phosphate binding can occur simultaneously and the dissociation of phosphate may actually promote the binding of carbamoyl phosphate for more efficient catalysis.

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