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Refinement and Comparisons of the Crystal Structures of Pig Cytosolic Aspartate Aminotransferase and Its Complex with 2-Methylaspartate*

Open AccessPublished:July 11, 1997DOI:https://doi.org/10.1074/jbc.272.28.17293
      Two high resolution crystal structures of cytosolic aspartate aminotransferase from pig heart provide additional insights into the stereochemical mechanism for ligand-induced conformational changes in this enzyme. Structures of the homodimeric native structure and its complex with the substrate analog 2-methylaspartate have been refined, respectively, with 1.74-Å x-ray diffraction data to an R value of 0.170, and with 1.6-Å data to an R value of 0.173. In the presence of 2-methylaspartate, one of the subunits (subunit 1) shows a ligand-induced conformational change that involves a large movement of the small domain (residues 12–49 and 327–412) to produce a “closed” conformation. No such transition is observed in the other subunit (subunit 2), because crystal lattice contacts lock it in an “open” conformation like that adopted by subunit 1 in the absence of substrate. By comparing the open and closed forms of cAspAT, we propose a stereochemical mechanism for the open-to-closed transition that involves the electrostatic neutralization of two active site arginine residues by the negative charges of the incoming substrate, a large change in the backbone (φ,ψ) conformational angles of two key glycine residues, and the entropy-driven burial of a stretch of hydrophobic residues on the N-terminal helix. The calculated free energy for the burial of this “hydrophobic plug” appears to be sufficient to serve as the driving force for domain closure.
      It frequently has been observed that an enzyme will undergo a large conformational change to bring catalytic groups into functionally active orientations in response to substrate binding at the active site. This type of ligand-induced conformational change was proposed by Koshland in his “induced fit” model (
      • Koshland Jr., D.E.
      ) and reflects the flexible nature of large proteins (
      • Huber R.
      ). X-ray crystallographic methods have been used to directly reveal ligand-induced structural changes in several enzymes (
      • Janin J.
      • Wodak S.J.
      ,
      • Bennett W.S.
      • Huber R.
      ); e.g. aspartate aminotransferase (see below), hexokinase (
      • Bennett Jr., W.S.
      • Steitz T.A.
      ), alcohol dehydrogenase (
      • Eklund H.
      • Samama J.-P.
      • Wallén L.
      • Brändén C.-I.
      • Åkeson Å
      • Jones T.A.
      ), and citrate synthase (
      • Remington S.
      • Wiegand G.
      • Huber R.
      ).
      Aspartate aminotransferase (AspAT)
      The abbreviations used are: AspAT, aspartate aminotransferase; cAspAT, cytosolic AspAT; mAspAT, mitochondrial AspAT; MeAsp, dl-2-methylaspartate; PEG, polyethylene glycol; r.m.s., root mean square.
      1The abbreviations used are: AspAT, aspartate aminotransferase; cAspAT, cytosolic AspAT; mAspAT, mitochondrial AspAT; MeAsp, dl-2-methylaspartate; PEG, polyethylene glycol; r.m.s., root mean square.
      is one of the key enzymes in amino acid metabolism. The enzyme is responsible for the following reversible transamination reaction.
      L­Aspartate+α­ketoglutarateL­glutamate+oxaloacetate


      REACTION1


      Oxaloacetate produced in this way in the cytosol can be converted to glucose via the gluconeogenesis pathway, or it can be used to indirectly transport NADH into the mitochondria via the malate-aspartate shuttle (
      • Dawson A.G.
      ). Since its discovery in 1937, AspAT has been studied extensively, and its functional, mechanistic, and structural properties have been reviewed in great detail (
      • Braunstein A.E.
      ,
      • Braunstein A.E.
      ,
      • Ivanov V.I.
      • Karpeisky M.Y.
      ,
      • Christen P.
      • Metzler D.E.
      ,
      • Jansonius J.N.
      • Vincent M.G.
      ). In early studies, the ligand-induced conformational changes of AspAT during catalysis were suggested by changes in the reactivity of a cysteine residue (
      • Birchmeier W.
      • Wilson K.J.
      • Christen P.
      ,
      • Gehring H.
      • Christen P.
      ). More recently, direct evidence for conformational changes upon ligation has been obtained from the x-ray crystallographic studies of AspAT from several sources: chicken mAspAT (
      • Jansonius J.N.
      • Vincent M.G.
      ,
      • McPhalen C.A.
      • Vincent M.G.
      • Picot D.
      • Jansonius J.N.
      • Lesk A.M.
      • Chothia C.
      ), chicken cAspAT (
      • Borisov V.V.
      • Borisova S.N.
      • Kachalova G.S.
      • Sosfenov N.I.
      • Vainshtein B.K.
      ,
      • Malashkevich V.N.
      • Strokopytov B.V.
      • Borisov V.V.
      • Dauter Z.
      • Wilson K.S.
      • Torchinsky Y.M.
      ), Escherichia coli AspAT (
      • Kamitori S.
      • Okamoto A.
      • Hirotsu K.
      • Higuchi T.
      • Kuramitsu S.
      • Kagamiyama H.
      • Matsuura Y.
      • Katsube Y.
      ,
      • Jäger J.
      • Moser M.
      • Sauder U.
      • Jansonius J.N.
      ) and pig cAspAT (
      • Arnone A.
      • Christen P.
      • Jansonius J.N.
      • Metzler D.E.
      ). In all cases, a similar open-to-closed transition has been observed. However, many aspects of the mechanism of the substrate-induced domain movement, such as the identification of all the residues that have an essential role in domain closure and the characterization of the driving force for domain movement, have yet to be fully resolved.
      Pig heart cAspAT is a dimeric enzyme of identical 412-residue subunits (molecular mass of 92,700 Da). Each subunit consists of a large and a small domain, and the active site is located at the interface between the two domains. At the active site, one molecule of the coenzyme pyridoxal 5′-phosphate is covalently linked to the ε-amino group of lysine 258 in the large domain through an aldimine linkage. Two different conformations for the small domain were characterized previously from a 2.7-Å resolution structure of native cAspAT and a 3.2-Å difference Fourier map of a cAspAT-substrate analog complex (
      • Arnone A.
      • Christen P.
      • Jansonius J.N.
      • Metzler D.E.
      ). In the absence of substrates, the two identical subunits are spatially related by 2-fold symmetry, adopting the so-called “open conformation.” The binding of substrate in the active site of one of the subunits (referred to as subunit 1) induces the small domain of that subunit to shift toward the active site, forming the “closed conformation.” In contrast to solution studies, which show that both subunits are reactive and independent (
      • Boettcher B.
      • Martinez-Carrion M.
      ,
      • Schlegel H.
      • Zaoralek P.E.
      • Christen P.
      ), only subunit 1 in crystalline cAspAT shows a conformational change on binding substrate because crystal lattice contacts lock the other subunit (subunit 2) in the open conformation (Fig. 1).
      Figure thumbnail gr8
      Figure 8Atom labeling convention and definition of torsional angles for the internal aldimine.
      Figure thumbnail gr1
      Figure 1Stereo diagram (drawn with MOLSCRIPT (
      • Kraulis P.J.
      )) showing an α-carbon tracing of the cAspAT dimer and the ligand-induced movement of the small domain that takes place when MeAsp binds to the active site of subunit 1 (solid thin lines). Crystal lattice contacts prevent the binding of MeAsp and small domain movement in subunit 2 (solid thick lines). Dashed lines show the position of the small domain of subunit 1 in the cAspAT-MeAsp complex. The positions of selected residues having structural or functional roles are labeled.
      Here we present the 1.74-Å structure of cAspAT and the 1.6-Å structure of cAspAT complexed with MeAsp. Analysis of these two structures reveals important elements of the stereochemical mechanism for domain movement in AspAT.

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