Structure of Escherichia coli glutamate decarboxylase (GADalpha) in complex with glutarate at 2.05 angstroms resolution.

Glutamate decarboxylase (GAD) is a pyridoxal enzyme that catalyzes the conversion of L-glutamate into gamma-aminobutyric acid and carbon dioxide. The Escherichia coli enzyme exists as two isozymes, referred to as GADalpha and GADbeta. Crystals of the complex of the recombinant isozyme GADalpha with glutarate as a substrate analogue were grown in space group R3, with unit-cell parameters a = b = 117.1, c = 196.4 angstroms. The structure of the enzyme was solved by the molecular-replacement method and refined at 2.05 angstroms resolution to an R factor of 15.1% (R(free) = 19.9%). The asymmetric unit contains a dimer consisting of two subunits of the enzyme related by a noncrystallographic twofold axis which is perpendicular to and intersects a crystallographic threefold axis. The dimers are related by a crystallographic threefold axis to form a hexamer. The active site of each subunit is formed by residues of the large domains of both subunits of the dimer. The coenzyme pyridoxal phosphate (PLP) forms an aldimine bond with Lys276. The glutarate molecule bound in the active site of the enzyme adopts two conformations with equal occupancies. One of the two carboxy groups of the glutarate occupies the same position in both conformations and forms hydrogen bonds with the N atom of the main chain of Phe63 and the side chain of Thr62 of one subunit and the side chains of Asp86 and Asn83 of the adjacent subunit of the dimer. Apparently, it is in this position that the distal carboxy group of the substrate would be bound by the enzyme, thus providing recognition of glutamic acid by the enzyme.

Hydrogel-based protein microchips: manufacturing, properties, and applications.

Here a simple, reproducible, and versatile method is described for manufacturing protein and ligand chips. The photo-induced copolymerization of acrylamide-based gel monomers with different probes (oligonucleotides, DNA, proteins, and low-molecular ligands) modified by the introduction of methacrylic groups takes place in drops on a glass or silicone surface. All probes are uniformly and chemically fixed with a high yield within the whole volume of hydrogel semispherical chip elements that are chemically attached to the surface. Purified enzymes, antibodies, antigens, and other proteins, as well as complex protein mixtures such as cell lysates, were immobilized on a chip. Avidin- and oligohistidine-tagged proteins can be immobilized within biotin- and Ni-nitrilotriacetic acid-modified gel elements. Most gel-immobilized proteins maintain their biological properties for at least six months. Fluorescence and chemiluminescence microscopy were used as efficient methods for the quantitative analysis of the microchips. Direct on-chip matrix-assisted laser desorption ionization-time of flight mass spectrometry was used for the qualitative identification of interacting molecules and to analyze tryptic peptides after the digestion of proteins in individual gel elements. We also demonstrate other useful properties of protein microchips and their application to proteomics and diagnostics.

[Study of the quaternary structure of glutamate carboxylase from Escherichia coli]. / Issledovanie chetvertichnoi struktury glutamatdekarboksilazy iz Escherichia coli.

It was shown by electron microscopy, that the native molecule of glutamate decarboxylase is a hexamer with dihedral symmetry; the subunits are situated at the apices of an octahedron. Apoenzyme at pH 6.0 is dissociated form. It were found s20.w - 12.8 +/- 0.54S and 5.51 +/- 0.43S for the native hexamer and a dissociated form, respectively. By column gel-filtration the molecular mass of the dissociated form was estimated as 105-106 kDa, this value corresponds to a dimer. There were 10 buried SH-groups per subunit in the hexamer, after dimer formation 8 of them became accessible. The reversible hexamer-dimer dissociation depends on pH and PLP. The pH dependences of the enzyme dissociation and activity are very similar. In the result of adding of 6 PLP equivalents to the dimers the reactivation and hexamer assembly were reached, the SH-groups burying preceded both these reactions. Effect of pH and PLP on the quaternary structure is known for some other PLP-enzymes. It may be the additional proof for the idea of a common ancestor for PLP-enzymes.

[Reactivation and reconstitution of glutamate decarboxylase upon the interaction of its dimers with pyridoxal phosphate]. / Reaktivatsiia i rekonstruktsiia geksamera glutamatdekarboksilazy pri vzaimodeistvii ee dimerov s piridoksal'fosfatom.

The relationship between the reactivation and reconstitution of the hexameric form of glutamate decarboxylase during the interaction of inactive apoenzyme dimers with pyridoxal phosphate (PLP) has been studied. It was shown that the restoration of enzymatic activity, appearance of spectral maximum at 340 nm, and reconstitution of the hexamer depend on the amount of PLP added; this reaction is completed when the PLP concentration reaches that of the initial enzyme. This native hexamer of the holo- and apoenzyme does not practically contain exposed sulfhydryl groups. Ten cysteine residues become available after DS-Na denaturation. The dimer of the apoenzyme contains 8 exposed and 2 buried cysteine residues. The hexamer formation from the dimers is accompanied by the burying of the cysteine residues. When half of the required PLP was added, 7 cysteine residues became buried in experiments with DTNB and six in experiments with 4.4'-DTDP. Further addition of PLP led to the disappearance of the exposed sulfhydryl groups.