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► Review of allostery of aspartate transcarbamoylase. ► Structural changes during the allosteric transition. ► Basis of allostery at the structural level.
The allosteric enzyme ...aspartate transcarbamoylase (ATCase) from Escherichia coli has been the subject of investigations for approximately 50years. This enzyme controls the rate of pyrimidine nucleotide biosynthesis by feedback inhibition, and helps to balance the pyrimidine and purine pools by competitive allosteric activation by ATP. The catalytic and regulatory components of the dodecameric enzyme can be separated and studied independently. Many of the properties of the enzyme follow the Monod, Wyman Changeux model of allosteric control thus E. coli ATCase has become the textbook example. This review will highlight kinetic, biophysical, and structural studies which have provided a molecular level understanding of how the allosteric nature of this enzyme regulates pyrimidine nucleotide biosynthesis.
The identification of a proper lead compound for fructose 1,6-bisphosphatase (FBPase) is a critical step in the process of developing novel therapeutics against type-2 diabetes. Herein, we have ...successfully generated a library of allosteric inhibitors against FBPase as potential anti-diabetic drugs, of which, the lead compound
1b was identified through utilizing a virtual high-throughput screening (vHTS) system, which we have developed. The thiazole-based core structure was synthesized via the condensation of α-bromo-ketones with thioureas and substituents on the two aryl rings were varied.
4c was found to inhibit pig kidney FBPase approximately fivefold better than
1b. In addition, we have also identified
10b, a tight binding fragment, which can be use for fragment-based drug design purposes.
Enzymes catalyze a particular reaction in cells, but only a few control the rate of this reaction and the metabolic pathway that follows. One specific mechanism for such enzymatic control of a ...metabolic pathway involves molecular feedback, whereby a metabolite further down the pathway acts at a unique site on the control enzyme to alter its activity allosterically. This regulation may be positive or negative (or both), depending upon the particular system. Another method of enzymatic control involves the cooperative binding of the substrate, which allows a large change in enzyme activity to emanate from only a small change in substrate concentration. Allosteric regulation and homotropic cooperativity are often known to involve significant conformational changes in the structure of the protein. Escherichia coli aspartate transcarbamoylase (ATCase) is the textbook example of an enzyme that regulates a metabolic pathway, namely, pyrimidine nucleotide biosynthesis, by feedback control and by the cooperative binding of the substrate, l-aspartate. The catalytic and regulatory mechanisms of this enzyme have been extensively studied. A series of X-ray crystal structures of the enzyme in the presence and absence of substrates, products, and analogues have provided details, at the molecular level, of the conformational changes that the enzyme undergoes as it shifts between its low-activity, low-affinity form (T state) to its high-activity, high-affinity form (R state). These structural data provide insights into not only how this enzyme catalyzes the reaction between l-aspartate and carbamoyl phosphate to form N-carbamoyl-l-aspartate and inorganic phosphate, but also how the allosteric effectors modulate this activity. In this Account, we summarize studies on the structure of the enzyme and describe how these structural data provide insights into the catalytic and regulatory mechanisms of the enzyme. The ATCase-catalyzed reaction is regulated by nucleotide binding some 60 Å from the active site, inducing structural alterations that modulate catalytic activity. The delineation of the structure and function in this particular model system will help in understanding the molecular basis of cooperativity and allosteric regulation in other systems as well.
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 Mg2+ concentration within the physiological range. In the presence of 2 mM divalent cations (Mg2+, Ca2+, Zn2+), 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.
Escherichia coli aspartate transcarbamoylase is feedback inhibited by CTP and UTP in the presence of CTP. Here, we show by X-ray crystallography that UTP binds to a unique site on each regulatory ...chain of the enzyme that is near but not overlapping with the known CTP site. These results bring into question all of the previously proposed mechanisms of allosteric regulation in aspartate transcarbamoylase.
Natural products often contain unusual scaffold structures that may be elaborated by combinatorial methods to develop new drug-like molecules. Visual inspection of more than 128 natural products with ...some type of anti-diabetic activity suggested that a subset might provide novel scaffolds for designing potent inhibitors against fructose 1,6-bisphosphatase (FBPase), an enzyme critical in the control of gluconeogenesis. Using
in silico docking methodology, these were evaluated to determine those that exhibited affinity for the AMP binding site. Achyrofuran from the South American plant
Achyrocline satureoides, was selected for further investigation. Using the achyrofuran scaffold, inhibitors against FBPase were developed. Compounds
15 and
16 inhibited human liver and pig kidney FBPases at IC
50 values comparable to that of AMP, the natural allosteric inhibitor.
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Escherichia coli aspartate transcarbamoylase (ATCase) allosterically regulates pyrimidine nucleotide biosynthesis. The enzyme is inhibited by CTP and can be further inhibited by UTP, although UTP ...alone has little or no influence on activity; however, the mechanism for the synergistic inhibition is still unknown. To determine how UTP is able to synergistically inhibit ATCase in the presence of CTP, we determined a series of X-ray structures of ATCase·nucleotide complexes. Analysis of the X-ray structures revealed that (1) CTP and dCTP bind in a very similar fashion, (2) UTP, in the presence of dCTP or CTP, binds at a site that does not overlap the CTP/dCTP site, and (3) the triphosphates of the two nucleotides are parallel to each other with a metal ion, in this case Mg2+, coordinated between the β- and γ-phosphates of the two nucleotides. Kinetic experiments showed that the presence of a metal ion such as Mg2+ is required for synergistic inhibition. Together, these results explain how the binding of UTP can enhance the binding of CTP and why UTP binds more tightly in the presence of CTP. A mechanism for the synergistic inhibition of ATCase is proposed in which the presence of UTP stabilizes the T state even more than CTP alone. These results also call into question many of the past kinetic and binding experiments with ATCase with nucleotides as the presence of metal contamination was not considered important.
Here, X-ray crystallography has been used to investigate the proposed double in-line displacement mechanism of
Escherichia coli alkaline phosphatase in which two of the three active-site metal ions ...have a direct role in catalysis. Two new X-ray crystal structures of the wild-type enzyme in the absence and presence of inorganic phosphate have been refined at 1.75 Å to final working
R-factors of 15.4 % and 16.4 %, respectively. In the refinement of both structures, residues in the active sites were treated anisotropically. The ellipsoids resulting from the partial anisotropic refinement show a clear route for the binding and release of substrate/product. In addition, a direct comparison of the refined structures with and without phosphate reveal a strong correlation between the occupancy of the third metal-binding site and the conformation of the Ser102 nucleophile. These findings clarify two important and unresolved aspects of the previously proposed catalytic mechanism, how Ser102 is activated for nucleophilic attack and why a magnesium ion in the third metal site is required for catalysis. Analysis of these results suggest that three metal-ion assisted catalysis is a more accurate description of the mechanism of the alkaline phosphatase reaction. A revised mechanism for the catalytic reaction of alkaline phosphatase is proposed on the basis of the two new X-ray crystal structures reported.
Many signaling and metabolic pathways rely on the ability of some of the proteins involved to undergo a substrate-induced transition between at least two structural states. Among the various models ...put forward to account for binding and activity curves of those allosteric proteins, the Monod, Wyman, and Changeux model for allostery theory has certainly been the most influential, although a central postulate, the preexisting equilibrium between the low-activity, low-affinity quaternary structure and the high-activity, high-affinity quaternary structure states in the absence of substrates, has long awaited direct experimental substantiation. Upon substrate binding, allosteric Escherichia coli aspartate transcarbamoylase adopts alternate quaternary structures, stabilized by a set of interdomain and intersubunit interactions, which are readily differentiated by their solution x-ray scattering curves. Disruption of a salt link, which is observed only in the low-activity, low-affinity quaternary structure, between Lys-143 of the regulatory chain and Asp-236 of the catalytic chain yields a mutant enzyme that is in a reversible equilibrium between at least two states in the absence of ligand, a major tenet of the Monod, Wyman, and Changeux model. By using this mutant as a magnifying glass of the structural effect of ligand binding, a comparative analysis of the binding of carbamoyl phosphate (CP) and analogs points out the crucial role of the amine group of CP in facilitating the transition toward the high-activity, high-affinity quaternary state. Thus, the cooperative binding of aspartate in aspartate transcarbamoylase appears to result from the combination of the preexisting quaternary structure equilibrium with local changes induced by CP binding.
The proposed double in-line displacement mechanism of
Escherichia coli alkaline phosphatase (AP) involving two-metal ion catalysis is based on NMR spectroscopic and X-ray crystallographic studies. ...This mechanism is further supported by the X-ray crystal structures of the covalent phospho-enzyme intermediate of the H331Q mutant AP and of the transition state complex between the wild-type enzyme and vanadate, a transition state analog. Kinetic and structural studies on several genetically engineered versions of AP illustrate the overall importance of the active site’s metal geometry, hydrogen bonding network and electrostatic potential in the catalytic mechanism.
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