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1.
Protein Sci ; 11(5): 1074-81, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-11967364

RESUMO

The available crystal structures of Escherichia coli aspartate transcarbamoylase (ATCase) show that the conserved residue Asp-162 from the catalytic chain interacts with essentially the same residues in both the T- and R-states. To study the role of Asp-162 in the regulatory properties of the enzyme, this residue has been replaced by alanine. The mutant D162A shows a 7700-fold reduction in the maximal observed specific activity, a twofold decrease in the affinity for aspartate, a loss of homotropic cooperativity, and decreased activation by the nucleotide effector adenosine triphosphate (ATP) compared with the wild-type enzyme. Small-angle X-ray scattering (SAXS) measurements reveal that the unliganded mutant enzyme adopts the T-quaternary structure of the wild-type enzyme. Most strikingly, the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) is unable to induce the T to R quaternary structural transition, causing only a small alteration of the scattering pattern. In contrast, addition of the activator ATP in the presence of PALA causes a significant increase in the scattering amplitude, indicating a large quaternary structural change, although the mutant does not entirely convert to the wild-type R structure. Attempts at modeling this new conformation using rigid body movements of the catalytic trimers and regulatory dimers did not yield a satisfactory solution. This indicates that intra- and/or interchain rearrangements resulting from the mutation bring about domain movements not accounted for in the simple model. Therefore, Asp-162 appears to play a crucial role in the cooperative structural transition and the heterotropic regulatory properties of ATCase.


Assuntos
Trifosfato de Adenosina/metabolismo , Aspartato Carbamoiltransferase/genética , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Ácido Fosfonoacéticos/análogos & derivados , Regulação Alostérica , Substituição de Aminoácidos , Aspartato Carbamoiltransferase/química , Aspartato Carbamoiltransferase/metabolismo , Escherichia coli , Ácido Fosfonoacéticos/metabolismo , Estrutura Quaternária de Proteína/efeitos dos fármacos , Especificidade por Substrato , Raios X
2.
Nat Struct Biol ; 8(5): 423-6, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11323717

RESUMO

Regulation of protein function, often achieved by allosteric mechanisms, is central to normal physiology and cellular processes. Although numerous models have been proposed to account for the cooperative binding of ligands to allosteric proteins and enzymes, direct structural support has been lacking. Here, we used a combination of X-ray crystallography and small angle X-ray scattering in solution to provide direct structural evidence that the binding of ligand to just one of the six active sites of Escherichia coli aspartate transcarbamoylase induces a concerted structural transition from the T to the R state.


Assuntos
Aspartato Carbamoiltransferase/química , Aspartato Carbamoiltransferase/metabolismo , Escherichia coli/enzimologia , Regulação Alostérica , Sítio Alostérico , Substituição de Aminoácidos/genética , Aspartato Carbamoiltransferase/genética , Domínio Catalítico , Cristalografia por Raios X , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Modelos Moleculares , Estrutura Quaternária de Proteína , Subunidades Proteicas , Difração de Raios X
3.
J Biol Chem ; 275(21): 15820-7, 2000 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-10748118

RESUMO

The genes from the thermophilic archaeabacterium Methanococcus jannaschii that code for the putative catalytic and regulatory chains of aspartate transcarbamoylase were expressed at high levels in Escherichia coli. Only the M. jannaschii PyrB (Mj-PyrB) gene product exhibited catalytic activity. A purification protocol was devised for the Mj-PyrB and M. jannaschii PyrI (Mj-PyrI) gene products. Molecular weight measurements of the Mj-PyrB and Mj-PyrI gene products revealed that the Mj-PyrB gene product is a trimer and the Mj-PyrI gene product is a dimer. Preliminary characterization of the aspartate transcarbamoylase from M. jannaschii cell-free extract revealed that the enzyme has a similar molecular weight to that of the E. coli holoenzyme. Kinetic analysis of the M. jannaschii aspartate transcarbamoylase from the cell-free extract indicates that the enzyme exhibited limited homotropic cooperativity and little if any regulatory properties. The purified Mj-catalytic trimer exhibited hyperbolic kinetics, with an activation energy similar to that observed for the E. coli catalytic trimer. Homology models of the Mj-PyrB and Mj-PyrI gene products were constructed based on the three-dimensional structures of the homologous E. coli proteins. The residues known to be critical for catalysis, regulation, and formation of the quaternary structure from the well characterized E. coli aspartate transcarbamoylase were compared.


Assuntos
Proteínas Arqueais/química , Aspartato Carbamoiltransferase/química , Mathanococcus/enzimologia , Sequência de Aminoácidos , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Cinética , Mathanococcus/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/química , Alinhamento de Sequência
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