Introduction The charge-density distribution in V20 a differs from that in Ti20 3 mainly with respect to the deformation of the metal atoms. The V atoms show a positive deformation of up to 0.1 e A -3 perpendicular to c in a plane containing three nearest V-atom sites across the edges of the surrounding O-atom octahedra, and a negative deformation of up to 0.3 e ]~-3 parallel to c between the nearest V-atom site across the faces of the O-atom octahedra. These observations are in accordance with theoretical band-structure calculations and confirm the existence of an e,, metal-metal bond which is directed across the common edges of the metalcentred O-atom octahedra. * Part I: Vincent, Yvon, Griittner & Ashkenazi (1980). t Present address: Biozentrum der Universitiit Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. 0567-7394/80/050808-06501.00 According to a study of the variation in the c/a ratio in corundum-type oxides (Goodenough, 1963, 1970, 1971; Prewitt, Shannon, Rogers & Sleight, 1969; McWhan, Rice & Remeika, 1969) and theoretical calculations (Ashkenazi & Weger, 1976; Ashkenazi & Chuchem, 1975; Castellani, Natoli & Ranninger, 1978), the metal-metal bonding in V20 3 is distinctly different from that in Ti20 3. Whereas the metal atoms in Ti203 interact mainly along the c direction through common faces of the O-atom octahedra, they interact in V20 3 mainly in directions perpendicular to the c direction through common edges of the O-atom octahedra (see metal-atom sites V, V' and V" in Fig. 1). In a previous article (Vincent, Yvon, Gr/ittner & Ashkenazi, 1980; hereafter referred to as VYGA), we have presented an experimentally determined electrondensity map for Ti20 3 which confirms the existence of a © 1980 International Union of Crystallography M. G. VINCENT, K. YVON AND J. ASHKENAZI 809 Ti--Ti bond along e. In the present study we report on the electron-density distribution in V20 3 and compare the bonding features found with those predicted by theory.

The X-ray crystal structures of three forms of the enzyme aspartate aminotransferase (EC 2.6.1.1) from chicken heart mitochondria have been refined by least-squares methods: holoenzyme with the co-factor pyridoxal-5′-phosphate bound at pH 7.5 (1.9 Å resolution), holoenzyme with pyridoxal-5′-phosphate bound at pH 5.1 (2.3 Å resolution) and holoenzyme with the co-factor pyridoxamine-5′-phosphate bound at pH 7.5 (2.2 Å resolution). The crystallographic agreement factors (R = ∑∥Fo¦−¦Fc∥∑¦Fo¦) for the structures are 0.166, 0.130 and 0.131, respectively, for all data in the resolution range from 10.0 Å to the limit of diffraction for each structure. The secondary, super-secondary and domain structures of the pyridoxal-phosphate holoenzyme at pH 7.5 are described in detail. The surface area of the interface between the monomer subunits of this dimeric α2 protein is unusually large, indicating a very stable dimer. This is consistent with biochemical data. Both subunit and domain interfaces are relatively smooth compared with other proteins. The interactions of the protein with its co-factor are described and compared among the three structures. Observed changes in co-factor conformation may be related to spectral changes and the energetics of the catalytic reaction. Small but significant adjustments of the protein to changes in co-factor conformation are seen. These adjustments may be accommodated by small rigid-body shifts of secondary structural elements, and by packing defects in the protein core.

The subunits of the dimeric enzyme aspartate aminotransferase have two domains: one large and one small. The active site lies in a cavity that is close to both the subunit interface and the interface between the two domains. On binding the substrate the domains close together. This closure completely buries the substrate in the active site and moves two arginine side-chains so they form salt bridges with carboxylate groups of the substrate. The salt bridges hold the substrate close to the pyridoxal 5'-phosphate cofactor and in the right position and orientation for the catalysis of the transamination reaction. We describe here the structural changes that produce the domain movements and the closure of the active site. Structural changes occur at the interface between the domains and within the small domain itself. On closure, the core of the small domain rotates by 13 degrees relative to the large domain. Two other regions of the small domain, which form part of the active site, move somewhat differently. A loop, residues 39 to 49, above the active site moves about 1 A less than the core of the small domain. A helix within the small domain forms the "door" of the active site. It moves with the core of the small domain and, in addition, shifts by 1.2 A, rotates by 10 degrees, and switches its first turn from the alpha to the 3(10) conformation. This results in the helix closing the active site. The domain movements are produced by a co-ordinated series of small changes. Within one subunit the polypeptide chain passes twice between the large and small domains. One link involves a peptide in an extended conformation. The second link is in the middle of a long helix that spans both domains. At the interface this helix is kinked and, on closure, the angle of the kink changes to accommodate the movement of the small domain. The interface between the domains is formed by 15 residues in the large domain packing against 12 residues in the small domain and the manner in which these residues pack is essentially the same in the open and closed structures. Domain movements involve changes in the main-chain and side-chain torsion angles in the residues on both sides of the interface. Most of these changes are small; only a few side-chains switch to new conformations.(ABSTRACT TRUNCATED AT 400 WORDS)

Single-crystal ultraviolet spectroscopy, X-ray absorption spectroscopy and EPR measurements have been used to examine the oxidation and oxygenation state of the dinuclear copper site of several types of hemocyanin crystals. The crystals contain Panulirus interruptus hemocyanin which forms hexameric molecules with a molecular mass of approximately 470 kDa. Three types of crystals have been investigated. Type-I monoclinic crystals, which have been used for the X-ray structure determination, contain virtually only deoxyhemocyanin. Type-II monoclinic crystals, which are less well ordered than the type-I crystals, contain a mixture of deoxy, oxy and met forms. Older crystals contain relatively more methemocyanin. A third, hexagonal, crystal form is also partially oxygenated, and, like the type-II monoclinic form, subject to gradual conversion to methemocyanin.

The pH dependence of 31P-NMR spectra of pig cytosolic aspartate aminotransferase, containing either N-(5'-phosphopyridoxyl)-L-aspartate or pyridoxal 5'-deoxymethylenephosphonate in place of the normal coenzyme pyridoxal 5'-phosphate, has been analysed. The chemical shifts of phosphopyridoxylaspartate and of pyridoxal 5'-deoxymethylenephosphonate model Schiff base in free solution show pK values of 6.3 and 7.4, attributable to the second deprotonation step of phosphate and phosphonate, respectively. However, these compounds behave very differently when bound to apoaspartate aminotransferase. 31P-NMR spectra of these enzyme derivatives indicate that the phosph(on)ate group remains dianionic throughout the pH range 4-8.5. A clear correlation between apparent pK values obtained from spectrophotometric titration of the coenzyme chromophore and those obtained by 31P NMR indicates that the same ionisation is being reported by both methods. The data are interpreted, on the basis of available crystallographic structures of chicken mitochondrial aspartate aminotransferase, to indicate that in each case the alteration in 31P chemical shift results from a conformational change in the coenzyme 5' side chain, in which one of the structures involves a near-eclipsed pair of bonds. Such a stressed conformation produces slight alterations in bond angles around the phosphorus atom, which in turn cause the observed change in 31P chemical shift. The evidence is taken to indicate that in this case 31P NMR is a sensitive reporter of stress in enzyme-bound pyridoxal 5'-phosphate and its derivatives.

The refined crystallographic structure of the "closed" conformation of chicken mitochondrial aspartate aminotransferase has been used as a template for the construction of models of the two Escherichia coli aminotransferases encoded by the tyrB and aspC genes. The main results are as follows: (1) Only minor changes are required in the coordinates of the backbone atoms to accommodate the large number of substituted side chains. (2) All deletions and insertions required to allow maximum primary sequence alignment are on the solvent-accessible surface. (3) Charged residues are all located on the surface, in contact with solvent, except for certain conserved active site residues. (4) The close packing within the hydrophobic core is maintained. (5) The interactions between the subunits are maintained. (6) Modeling of tyrosine as an external aldimine into the active sites points to several residues that could be involved in determining the substrate specificities of these aminotransferases.

Phosphoserine aminotransferase from E.coli has been crystallized in space group P212121, with one ∝2 dimeric molecule per asymmetric unit. Single crystal microspectrophotometric measurements have shown that the enzyme is catalytically active in the crystal. X-Ray data for the native form and 2 heavy atom derivatives have been collected on a CAD4-di f fractometer. A 6Å resolution Fourier-map has been calculated? interpretation is underway. The orientation of the noncrystallographic two-fold axis of the dimer has been determined. Solution studies have shown that the activity maximum is below the pK of the internal aldimine. The quaternary and secondary structure have been determined by analytical ultracentrifugation and CD measurements.

N-(5'-Phosphoribosyl)anthranilate isomerase-indole-3-glycerol-phosphate synthase from Escherichia coli is a monomeric bifunctional enzyme of Mr 49,500 that catalyzes two sequential reactions in the biosynthesis of tryptophan. The three-dimensional structure of the enzyme has been determined at 2.8-A resolution by x-ray crystallography. The two catalytic activities reside on distinct functional domains of similar folding, that of an eightfold parallel beta-barrel with alpha-helices on the outside connecting the beta-strands. Both active sites were located with an iodinated substrate analogue and found to be in depressions on the surface of the domains created by the outward-curving loops between the carboxyl termini of the beta-sheet strands and the subsequent alpha-helices. They do not face each other, making "channeling" of the substrate between active sites virtually impossible. Despite the structural similarity of the two domains, no significant sequence homology was found when topologically equivalent residues were compared.

The 31P chemical shift of pyridoxal phosphate (PLP) in native cytosolic aspartate aminotransferase (cAAT) has been reported to be pH dependent with a pK of 6.2 (Schnackerz, 1984) even though X-ray data suggest that the cofactor phosphate group remains dianionic throughout. For further information on the pH dependence of 31P NMR spectra apo-cAAT reconstituted with phosphopyridoxyl aspartate and pyridoxal 5′deoxymethylene-phosphonate was measured. The chemical shifts for the two cofactor analogues when bound to apo-cAAT were found to be pH independent and should correspond to the phosph(on)ate dianion. Thus, the 31P NMR data on native cAAT can only be interpreted as the protonation/deprotonation equilibrium of the PLP-Lys 258 "internal aldimine" (pK=6.2). It is proposed that protonation of the aldimine exerts strain in the 5′-phosphate ester linkage, resulting in modified O-P-O bond angles which in turn cause changes in the 31P chemical shift.

Glutamate decarboxylase was purified from Escherichia coli and crystallized by vapour diffusion and microdialysis techniques in the presence of polyethylene glycol. Growth of large crystals suitable for X-ray studies is particularly favoured by low concentrations of citric or glutaric acid, which are both effective inhibitors of the enzyme. Inhibitor binding must induce the conformational changes that are essential for crystal nucleation since no crystallization occurred with non-inhibitory buffers of similar ionic composition. The crystals, of space group R3, diffract to a resolution of 2.9 Ȧ. The dimensions of the rhombohedral unit cell are a = b = c = 116 Ȧ, α= β = γ =116° with a dimer in the asymmetric unit. The equivalent hexagonal cell has a = b = 199 Ȧ, c = 69 Ȧ, α= β = 90°, γ= 120°.

Inspection of the structural models of five refined crystal structures of different unliganded and liganded forms of mitochondrial aspartate aminotransferase (McPhalen et al.P this Volume) has led to a number of observations about ligand binding and conformational changes involving both the "small domain" and the coenzyme that are relevant to the catalytic mechanism of this enzyme.

Ornithine aminotransferase was purified from rat liver and crystallized in the presence of ammonium sulphate and poly(ethylene glycol) (PEG 4000). The crystallographic threefold symmetry observed for the resulting two crystal forms stimulated a re-examination of the enzyme's quaternary structure in solution by analytical ultracentrifugation and chemical cross-linking. The results indicate that the oligomeric state or ornithine aminotransferase, under conditions similar to those used in crystallization experiments, is a hexamer (Mr = 256,000) rather than a tetramer or higher oligomers as reported previously. The subunits of the enzyme are identical (Mr = 45,000). Only the hexagonal prismatic crystals obtained with PEG 4000 were suitable for crystallographic studies and diffracted X-rays to a resolution of at least 0.16 nm. However, these crystals contained an unusual element of disorder which was persistent under a variety of conditions and was only noticeably diminished in the presence of the non-ionic detergent octyl beta-glucoside. The crystals apparently belong to the trigonal space group P3(1)12 (or enantiomorph) with axial lengths of a = 19.5 nm, c = 5.9 nm and contain three monomers per asymmetric unit.

The 3-dimensional structures of five forms of the mitochondrial aspartate aminotransferase (mAAT) from chicken heart have been solved by X-ray crystallography. The goal of our work is to understand the catalytic mechanism of AAT by studying the structures of analogs of catalytic intermediates. Comparisons among the structures confirm previous observations of gross conformational changes during catalysis. More subtle differences are seen in several regions of the protein, including the active site, based on the refined higher-resolution structures.

Conference Article| June 01 1984 Three-dimensional structure of mitochondrial aspartate aminotransferase and some functional derivatives: implications for its mode of action JOHAN N. JANSONIUS; JOHAN N. JANSONIUS *Biozentrum, Universität Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for other works by this author on: This Site PubMed Google Scholar GREGOR EICHELE; GREGOR EICHELE *Biozentrum, Universität Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for other works by this author on: This Site PubMed Google Scholar GEOFFREY C. FORD; GEOFFREY C. FORD *Biozentrum, Universität Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for other works by this author on: This Site PubMed Google Scholar JACK F. KIRSCH; JACK F. KIRSCH *Biozentrum, Universität Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for other works by this author on: This Site PubMed Google Scholar DANIEL PICOT; DANIEL PICOT *Biozentrum, Universität Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for other works by this author on: This Site PubMed Google Scholar CHRISTINA THALLER; CHRISTINA THALLER *Biozentrum, Universität Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for other works by this author on: This Site PubMed Google Scholar MICHAEL G. VINCENT; MICHAEL G. VINCENT *Biozentrum, Universität Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for other works by this author on: This Site PubMed Google Scholar HEINZ GEHRING; HEINZ GEHRING †Biochemisches Institut der Universität Zurich, Zürichbergstrasse 4, CH-8028 Zürich, Switzerland Search for other works by this author on: This Site PubMed Google Scholar PHILIPP CHRISTEN PHILIPP CHRISTEN †Biochemisches Institut der Universität Zurich, Zürichbergstrasse 4, CH-8028 Zürich, Switzerland Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1984) 12 (3): 424–427. https://doi.org/10.1042/bst0120424 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation JOHAN N. JANSONIUS, GREGOR EICHELE, GEOFFREY C. FORD, JACK F. KIRSCH, DANIEL PICOT, CHRISTINA THALLER, MICHAEL G. VINCENT, HEINZ GEHRING, PHILIPP CHRISTEN; Three-dimensional structure of mitochondrial aspartate aminotransferase and some functional derivatives: implications for its mode of action. Biochem Soc Trans 1 June 1984; 12 (3): 424–427. doi: https://doi.org/10.1042/bst0120424 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1984 Biochemical Society1984 Article PDF first page preview Close Modal You do not currently have access to this content.

Aspartate aminotransferase is a pyridoxal phosphate-dependent enzyme that catalyses the transamination reaction: l-aspartate + 2-oxoglutarate ⇋ oxaloacetate + l-glutamate. The enzyme shuttles between its pyridoxal and pyridoxamine forms in a double-displacement process. This paper proposes a mechanism of action that delineates the dynamic role of the protein moiety of this enzyme. It is based on crystallographically determined spatial structures (at 2.8 Å resolution) of the mitochondrial isoenzyme in its unliganded forms and in complexes with substrate analogues, as well as on model building studies.

A method for determining the polarization factor for a repeatedly reflected X-ray beam is described. It is shown that, provided the relative orientations of the pre-specimen reflectors are restricted to special geometries (the usual cases), the appropriate expression for an unpolarized beam reflected m times can be simply derived. The treatment is extended to a plane-polarized beam, resulting in an expression dependent on polarization effects from the specimen alone and hence independent of the state of perfection of crystal monochromators. The latter expression may have some relevance to experiments performed with synchrotron radiation.