Structural basis for ordered substrate binding and cooperativity in aspartate transcarbamoylase

Abstract

X-ray structures of aspartate transcarbamoylase in the absence and presence of the first substrate carbamoyl phosphate are reported. These two structures in conjunction with in silico docking experiments provide snapshots of critical events in the function of the enzyme. The ordered substrate binding, observed experimentally, can now be structurally explained by a conformational change induced upon the binding of carbamoyl phosphate. This induced fit dramatically alters the electrostatics of the active site, creating a binding pocket for aspartate. Upon aspartate binding, a further change in electrostatics causes a second induced fit, the domain closure. This domain closure acts as a clamp that both facilitates catalysis by approximation and also initiates the global conformational change that manifests homotropic cooperativity.allosteric transition ͉ induced fit ͉ homotropic cooperativity E nzymes that exhibit positive cooperativity play a vital role in the regulation of the rates of key metabolic pathways by amplifying the response of a pathway to an effector molecule. In the case of aspartate transcarbamoylase (ATCase), substrateinduced domain closure triggers a quaternary conformational change that results in the observed homotropic cooperativity, one mechanism by which this enzyme controls the rate of de novo pyrimidine biosynthesis. Understanding the molecular features of domain closure not only provides insights into the mechanism of homotropic cooperativity but also demonstrates how ligandinduced domain closure can be used as part of catalytic mechanism of many enzymes (1, 2).The Escherichia coli ATCase 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. The three active sites in the catalytic subunit are shared across the interface between adjacent chains (3, 4), whereas the regulatory subunits contain the binding sites for the heterotropic activator, ATP, as well as the heterotropic inhibitors, CTP and UTP. Each catalytic chain is composed of two structural domains, the carbamoyl phosphate (CP) domain (residues 1-135 and 292-310) and the L-aspartate (Asp) domain (residues 136-291), which contain the binding sites for CP and Asp, respectively. Each regulatory chain is also composed of two structural domains, the AL domain (residues 1-100) and the Zn domain (residues 101-153), which contain the binding sites for the allosteric effectors and the structural Zn, respectively. In mammals ATCase exists as a component of the multienzyme complex CAD (carbamoyl phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase) (5), and in humans, CAD has become a target for the development of antiproliferation drugs (6). The E. coli enzyme and the ATCase portion of CAD are 44% conserved, and all residues known to be involved in substrate binding and catalysis are conserved. Domain closure is triggered by the ordered binding of the substrates (7), with CP binding before Asp, and N-carbamoyl-L-...

Direct observation in solution of a preexisting structural equilibrium for a mutant of the allosteric aspartate transcarbamoylase

“…Indeed, according to the linkage equations of Wyman (35,36), it follows that aspartate and its analogs have higher affinity for the T state than for the R state of ATCase, a result difficult to reconcile with the fact that the wild-type enzyme exhibits cooperative aspartate binding. In a recently reported in silico docking study, CP and aspartate were found to bind in strongly overlapping positions within the active site of the unliganded wild-type enzyme (13). Only in the presence of CP does aspartate bind productively.…”

“…Two functional and structural states of the enzyme have been characterized by a variety of techniques: a less active, low-affinity, T-state, stabilized by CTP and a more active, high-affinity, R-state stabilized by the substrates or the bisubstrate analog Nphosphonacetyl-L-aspartate (PALA). The corresponding crystal structures (11,12) recently refined (13,14) described the T-to-R transition as an 11-Å (1 Å ϭ 0.1 nm) expansion along the 3-fold axis together with subunit and domain rotations (15) resulting in disruption of the r1-c4 interface between regulatory and catalytic chains. This quaternary rearrangement is accompanied by tertiary changes in both the catalytic and regulatory chains, most notably the 80s and 240s loops of the catalytic chains.…”

Structural Model of the R State of Escherichia coli Aspartate Transcarbamoylase with Substrates Bound

“…15 The molecular mechanism responsible for the ordered binding of Asp, which takes place only after CP binds in the active site has been proposed based upon structures of ATCase in the absence and presence of CP. 13 The binding of CP induces local conformational changes resulting in the formation of a new highly electropositive binding pocket for Asp. 13 To understand the binding of Asp to the enzyme•CP complex and the concurrent conformational changes induced by Asp binding, X-ray crystallography was employed.…”

“…13 The binding of CP induces local conformational changes resulting in the formation of a new highly electropositive binding pocket for Asp. 13 To understand the binding of Asp to the enzyme•CP complex and the concurrent conformational changes induced by Asp binding, X-ray crystallography was employed. We were able to obtain data quality crystals of the R state of the enzyme with substrates bound by utilized a mutant version of the enzyme with the D236A mutation.…”

“…An T-state X-ray structure of the enzyme•CP complex has revealed the molecular details utilized by the enzyme to achieve an ordered-binding mechanism by creating the Asp binding site only after CP binds. 13 However, no R-state structure of the enzyme with substrates bound immediately before the nucleophilic attack has been determined. In order to obtain this structure we utilized a mutant version of ATCase (D236A) in which one critical T-state interaction, between Asp236 on the catalytic chain and Lys143 on the regulatory chain, was eliminated.…”