E. coli Aspartate Transcarbamoylase (ATCase)

Allen Sanderlin '15 and Kaitlin Creamer '16


I. Introduction

Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate (CP) to produce N-carbamoyl-L-aspartate (CAA). This reaction is the first committed step of the pyrimidine biosynthetic pathway.

Cytidine triphosphate (CTP) and uridine triphosphate (UTP) are two end-products of pyrimidine biosynthesis. These compounds serve as cofactors in several metabolic reactions and are substrates in RNA synthesis. Subsequent modifications of these nucleotide triphosphates (NTPs) generate deoxy analogs for use in DNA synthesis.

In mammals, ATCase forms part of the multi-enzyme complex CAD (alongside carbamoyl phosphate synthetase II and dihydroorotase) that catalyzes several steps in pyrimidine biosynthesis. Since pyrimidines are essential for DNA synthesis during cell division, ATCase inhibitors such as the transition-state analogue N-(phosphonacetyl)-L-aspartate (PALA) have the potential for slowing tumor growth in cancer.

Allosteric regulation of ATCase by CTP and UTP prevents a pyrimidine surplus. These effectors synergistically inhibit ATCase activity, establishing a negative feedback loop. Conversely, adenosine triphosphate (ATP) and guanosine triphosphate (GTP) - both end-products of the parallel purine biosynthetic pathway - can act together to stimulate ATCase activity. Ultimately, the interplay between NTPs and ATCase ensures an appropriate balance of purines and pyrimidines within the cell. 

II. General Structure

The Escherichia coli ATCase is a 310 kDa dodecamer consisting of two catalytic homotrimers and three regulatory homodimers encoded by pyrB and pyrI, respectively. The interfaces between adjacent catalytic subunits form six active sites for binding of CP and L-aspartate while the interfaces between adjacent regulatory subunits form six allosteric sites for NTP binding . Each regulatory subunit contains a zinc-binding domain that is necessary for assembly of the complex. Within this domain, a Zn2+ cation is tetrahedrally coordinated to Cys109, Cys114, Cys138, and Cys141 . Each catalytic subunit contains eleven parallel beta sheets and eleven alpha helices that alternate to form a beta-alpha-beta motif . Each regulatory subunit contains nine antiparallel beta sheets and three alpha helices ; four of these sheets form a beta-meander motif in the aforementioned zinc-binding domain. 

III. Conformational Changes

CP and L-aspartate bind sequentially to an active site at the interface between adjacent catalytic subunits. Substrate binding induces conformational changes that close the active site and bring the substrates together for catalysis. Furthermore, these conformational changes near the active site are accompanied by a global conformational change: ATCase transitions from a low activity, low affinity T ("tense") state to a high activity, high affinity R ("relaxed") state. These states coexist within a dynamic equilibrium that is sensitive to substrate availability and allosteric regulation. During this transition, the regulatory dimers rotate by 15 and strengthen their interface , the catalytic trimers rotate by 12 to nearly eclipse each other , and the complex expands 11 to enlarge the solvent-exposed central cavity leading to the active sites . Formation of CAA allows the active sites to reopen for product release and new substrate binding, facilitated by the increased solvent exposure of the active sites in the R state. While CP and L-aspartate are abundant, ATCase remains in the R state. Once the substrates are exhausted, the enzyme returns to the T state. 

IV. Catalysis

At the interface between two adjacent catalytic subunits, the nucleophilic amino group of L-aspartate attacks the phosphoanhydride carbonyl carbon of CP, displacing the phosphate leaving group. Several residues in the electropositive active site stabilize the negatively-charged transition state of the condensation reaction with greater binding strength than for the substrates or products. The phosphate group is neutralized by interactions with the side-chains of Ser52, Arg54, and Arg105; the backbones of Thr53, Arg54, and Thr55; and the side-chains of Ser80 and Lys84 donated by an adjacent catalytic subunit . The amino group attached to the tetrahedral carbon contacts the side-chain of Gln137 while the negatively-charged oxygen attached to the tetrahedral carbon is neutralized by interactions with the side-chains of Arg105 and His134 . Finally, the amide nitrogen is stabilized by contacting the backbone of Leu267 while the alpha- and beta-carboxylates are neutralized by interactions with the side-chains of Lys84, Arg167, Arg229, and Gln231 .  

V. Regulation

ATCase is allosterically regulated by NTPs binding to the interface between adjacent regulatory subunits. Each allosteric site contains two nucleotide-specific subsites, A and B, that each bind one NTP and are bridged by a Mg2+ cation (or another divalent cation like Zn2+ or Ca2+). The A subsite preferentially binds CTP or ATP and the B subsite preferentially binds UTP or ATP. Subsite specificity for GTP is currently unknown. The bridging Mg2+ is coordinated to two water molecules and to the beta- and gamma-phosphate groups of each NTP in the allosteric site . In the next button, warm colors indicate regions of stronger intermolecular interactions while cool colors indicate sites of weaker interactions .

Within the A subsite, the phosphate groups are neutralized by interactions with the side-chains of His20 and Lys94; the ribose is stabilized by contacts with the side-chains of Asp19 and Lys60; and the nitrogen ring base is stabilized by interactions with the side-chain of Lys60 and the backbones of Ile12 and Tyr89. . The 4-keto group of UTP is incompatible for hydrogen-bonding with the backbone carbonyls, leaving CTP and ATP to compete for binding to the A subsite. Within the B subsite, the phosphate groups are neutralized by interactions with the side-chains of His20, Ser50, and Lys56, and with the backbone of Glu52. The nitrogen ring base is stabilized by contacting the side-chain of Lys60. The 4-amino group of CTP is electrostatically repulsed by the side-chain of Lys60, leaving UTP and ATP to compete for binding to the B subsite .

Although NTP binding induces minor conformational changes in the catalytic subunits, it has a much greater effect on the N-termini of the regulatory subunits. With CTP and UTP bound, the N-termini curl inward and the interface between adjacent subunits is not stabilized, thereby disrupting the R state. In this conformation, the N-terminal backbones of Gln8 and Val9 contact the pyrimidine ring of UTP in the B subsite of the same regulatory subunit . Alternatively, the large purine rings of ATP and GTP can push the N-termini into the B subsites of the opposing regulatory subunits, thereby locking the interface and favoring the R state. Upon crossing over, the N-terminal Lys6 side-chain can neutralize the NTP phosphate groups in both subsites of the opposing regulatory subunit . Glu52 and Tyr89 are included in the above two buttons for reference.

CTP and UTP inhibit ATCase by weakening the R state; conversely, ATP and GTP activate ATCase by strengthening the R state. Although these allosteric effectors cannot induce a global conformational change, they can shift the equilibrium between the R and T states. Ultimately, this dynamic equilibrium ensures an appropriate balance of purines and pyrimidines within the cell.

VI. References

Cockrell GM, Zheng Y, Guo W, Peterson AW, Truong JK, and Kantrowitz ER. 2013. New paradigm for allosteric regulation of Escherichia coli aspartate transcarbamoylase. Biochemistry 52: 8036-8047.

Honzatko RB, Crawford JL, Monaco HL, Ladner JE, Edwards BFP, Evans DR, Warren SG, Wiley DC, Ladner RC, and Lipscomb WN. 1982. Crystal and molecular structures of native and CTP-liganded aspartate carbamoyltransferase from Escherichia coli. J Mol Biol 160: 219-263.

Jin L, Stec B, Lipscomb WN, and Katrowitz ER. 1999. Insights into the mechanisms of catalysis and heterotropic regulation of Escherichia coli aspartate transcarbamoylase based upon a structure of the enzyme complexed with the bisubstrate analogue N-phosphonacetyl-L-aspartate at 2.1-. Proteins 37: 729-742.

Kantrowitz ER. 2012. Allostery and cooperativity in Escherichia coli aspartate transcarbamoylase. Arch Biochem Biophys 519: 81-90.

Lipscomb WN and Kantrowitz ER. 2012. Structure and mechanisms of Escherichia coli aspartate transcarbamoylase. Acc Chem Res 45: 444-453.

Peterson AW, Cockrell GM, and Kantrowitz ER. 2012. A second allosteric site in Escherichia coli aspartate transcarbamoylase. Biochemistry 51: 4776-4778.

Stevens RC, Gouaux JE, and Lipscomb WN. 1990. Structural consequences of effector binding to the T state of aspartate carbamoyltransferase: crystal structures of the unligated and ATP- and CTP-complexed enzymes at 2.6- resolution. Biochemistry 29: 7691-7701.

Wang J, Stieglitz KA, Cardia JP, and Kantrowitz ER. 2005. Structural basis for ordered substrate binding and cooperativity in aspartate transcarbamoylase. PNAS 102: 8881-8886.

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