Structure of the E. coli Aspartate Transcarbamoylase Trapped in the Middle of the Catalytic Cycle Kimberly A. Stieglitz 1 , Kelly J. Dusinberre 1 , James P. Cardia 1 Hiro Tsuruta 2 and Evan R. Kantrowitz 1 * 1 Department of Chemistry Merkert Chemistry Center Boston College, Chestnut Hill MA 02467, USA 2 Stanford Synchrotron Radiation Laboratory, Stanford Linear Accelerator Center MS69, 2575 Sand Hill Rd Menlo Park, CA 94025, USA Snapshots of the catalytic cycle of the allosteric enzyme aspartate transcarbamoylase have been obtained via X-ray crystallography. The enzyme in the high-activity high-affinity R state contains two catalytic chains in the asymmetric unit that are different. The active site in one chain is empty, while the active site in the other chain contains an analog of the first substrate to bind in the ordered mechanism of the reaction. Small angle X-ray scattering shows that once the enzyme is converted to the R state, by substrate binding, the enzyme remains in the R state until substrates are exhausted. Thus, this structure represents the active form of the enzyme trapped at two different stages in the catalytic cycle, before the substrates bind (or after the products are released), and after the first substrate binds. Opening and closing of the catalytic chain domains explains how the catalytic cycle occurs while the enzyme remains globally in the R-quaternary structure. q 2005 Published by Elsevier Ltd. Keywords: allosteric enzyme; small-angle X-ray scattering; cooperativity; enzyme mechanism *Corresponding author Introduction Escherichia coli aspartate transcarbamoylase (EC 2.1.3.2; ATCase) catalyzes the first step in pyrimi- dine biosynthesis, the condensation of carbamoyl phosphate (CP) and aspartate (Asp) to form N-carbamoyl-L-aspartate (CA) and inorganic phos- phate (P i ). This enzyme controls the rate of pyrimidine metabolism by homotropic and hetero- tropic interactions. The holoenzyme is composed of six chains (M r 34,000 each) grouped into two trimeric catalytic subunits, and six chains (M r 17,000 each) grouped into three dimeric regulatory subunits. Each catalytic chain is composed of CP and Asp domains that are primarily responsible for the binding of the two substrates CP and Asp, respectively. Each of the three active sites in the catalytic subunit are composed of residues from both the CP and Asp domains as well as residues of the 80s loop from an adjacent catalytic chain. 1,2 Each regulatory chain is composed of the Al domain that contains the allosteric effector binding site, and the Zn domain that contains a structural Zn site. The regulatory subunits contain the binding sites for the heterotropic activator, ATP, as well as the hetero- tropic inhibitors, CTP and UTP. The reaction follows an ordered mechanism with CP binding before Asp, and CA leaving before P i . 3 When CP binds to the enzyme, local conformational changes occur within the CP domain of the catalytic chain causing a change in the electrostatic environment of the active site, creating the binding pocket for Asp. 4 The binding of Asp to the E$CP complex induces a domain closure in the catalytic chains, which in turn induces the dramatic quaternary structural change from the T to the R structure, involving an 0022-2836/$ - see front matter q 2005 Published by Elsevier Ltd. Abbreviations used: ATCase, E. coli aspartate transcar- bamoylase holoenzyme; C1/C6, the two catalytic chains of ATCase in the asymmetric unit of the crystal; R1/R6, the two regulatory chains of ATCase in the asymmetric unit of the crystal; SAXS, small angle X-ray scattering; PALA, N-phosphonacetyl-L-aspartate; PAM, phospho- noacetamide; D236Ac, the mutant ATCase in which Asp236 in the catalytic chains have been replaced by Ala; K143Ar, the mutant ATCase in which Lys143 in the regulatory chains have been replaced by Ala; T PAM , the X-ray structure of ATCase in the presence of PAM (PDB code: 3AT1); T CP , the X-ray structure of ATCase in the presence of CP (PDB code: 1ZA2); R PM , the X-ray structure of ATCase in the presence of PAM and malonate (PDB code: 8AT1); R PALA , the X-ray structure of ATCase in the presence of PALA (PDB code: 1D09); R 236_PAM , the X-ray structure of the D236Ac ATCase in the presence of PAM determined here. E-mail address of the corresponding author: evan.kantrowitz@bc.edu doi:10.1016/j.jmb.2005.07.046 J. Mol. Biol. (2005) 352, 478–486