Arg1098 is critical for the chloride dependence of human angiotensin I-converting enzyme C-domain catalytic activity

Angiotensin (Ang) I-converting enzyme (ACE) is a Zn²+ metalloprotease with two homologous catalytic domains. Both the N- and C-terminal domains are peptidyl dipeptidases. Hydrolysis by ACE of its decapeptide substrate Ang I is increased by Cl−, but the molecular mechanism of this regulation is unclear. A search for single substitutions to Gln among all conserved basic residues (Lys/Arg) in human ACE C-domain identified R1098Q as the sole mutant that lacked Cl− dependence. Cl−dependence is also lost when the equivalent Arg in the N-domain, Arg⁵⁰⁰, is substituted with Gln. The Arg¹⁰⁹⁸ to Lys substitution reduced Cl− binding affinity by ∼100-fold. In the absence of Cl−, substrate binding affinity (1/K m) of and catalytic efficiency (k cat/K m) for Ang I hydrolysis are increased 6.9- and 32-fold, respectively, by the Arg¹⁰⁹⁸ to Gln substitution, and are similar (<2-fold difference) to the respective wild-type C-domain catalytic constants in the presence of optimal [Cl−]. The Arg¹⁰⁹⁸ to Gln substitution also eliminates Cl− dependence for hydrolysis of tetrapeptide substrates, but activity toward these substrates is similar to that of the wild-type C-domain in the absence of Cl−. These findings indicate that: 1) Arg¹⁰⁹⁸ is a critical residue of the C-domain Cl−-binding site and 2) a basic side chain is necessary for Cl− dependence. For tetrapeptide substrates, the inability of R1098Q to recreate the high affinity state generated by the Cl−-C-domain interaction suggests that substrate interactions with the enzyme-bound Cl− are much more important for the hydrolysis of short substrates than for Ang I. Since Cl− concentrations are saturating under physiological conditions and Arg¹⁰⁹⁸ is not critical for Ang I hydrolysis, we speculate that the evolutionary pressure for the maintenance of the Cl−-binding site is its ability to allow cleavage of short cognate peptide substrates at high catalytic efficiencies.

Changes in zinc ligation promote remodeling of the active site in the zinc hydrolase superfamily

The zinc hydrolase superfamily is a group of divergently related proteins that are predominantly enzymes with a zinc-based catalytic mechanism. The common structural scaffold of the superfamily consists of an eight-stranded β-sheet flanked by six α-helices. Previous analyses, while acknowledging the likely divergent origins of leucine aminopeptidase, carboxypeptidase A and the co-catalytic enzymes of the metallopeptidase H clan based on their structural scaffolds, have failed to find any homology between the active sites in leucine aminopeptidase and the metallopeptidase H clan enzymes. Here we show that these two groups of co-catalytic enzymes have overlapping dizinc centers where one of the two zinc atoms is conserved in each group. Carboxypeptidase A and leucine aminopeptidase, on the other hand, no longer share any homologous zinc-binding sites. At least three catalytic zinc-binding sites have existed in the structural scaffold over the period of history defined by available structures. Comparison of enzyme-inhibitor complexes show that major remodeling of the substrate-binding site has occurred in association with each change in zinc ligation in the binding site. These changes involve re-registration and re-orientation of the substrate. Some residues important to the catalytic mechanism are not conserved amongst members. We discuss how molecules acting in trans may have facilitated the mutation of catalytically important residues in the active site in this group.

A despecialization step underlying evolution of a family of serine proteases

In the trypsin superfamily of serine proteases, non-trypsin-like primary specificities have arisen in only two monophyletic descendent subbranches. We have recreated an ancestor to one of these subbranches (granzyme) using phylogenetic inference, gene synthesis, and protein expression. This ancestor has two unusual properties. First, it has broad primary specificity encompassing the entire repertoire of novel primary specificities found in its descendents. Second, unlike extant members that have narrow primary specificities, the ancestor exhibits tolerance to mutational changes in primary specificity-conferring residues—that is, structural plasticity. Molecular modeling and mutagenesis studies indicate that these unusual properties are due to a particularly wide substrate binding pocket. These two crucial properties of the ancestor not only distinguish it from its extant descendents but also from the trypsin-like proteases that preceded it. This indicates that a despecialization step, characterized by broad specificity and structural plasticity, underlies evolution of new primary specificities in this protease superfamily.

Suggestions regarding the principles which may be adopted by the Caribbean countries in negotiating agreements for the avoidance of double taxation with developed countries

Includes bibliography

A contradição entre herança ibérica e modernização brasileira no pensamento político de Sérgio Buarque de Holanda: o livro Raízes do Brasil

Pós-graduação em História - FCLAS

Produtividade e composição bromatológica do capim-Tobiatã em função da adubação NPK

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Paleoecologia e tafonomia da floresta petrificada do Tocantins setentrional (Bacia do Parnaíba, Permiano)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Segurança na América Latina: cenários e perspectivas

In the post Cold War period, in the security field, has increased the mechanisms of cooperation and coordination among the Latin American states, in the context of so called confidence-building measures. These arrangements presented as important and efficient tools of political-strategic articulation and assist in the maintenance of peace in the region. The paper presents some considerations about the current scenario in terms of security and defense in Latin America. Initially, presents concepts of security and defense and perceptions of threats by the states. Then, discusses the mechanisms of cooperation and coordination in security have been established on the American continent in the periods of the Cold War and post Cold War. Finally, presents considerations regarding the scenario and the prospects of security issues in the region.

Culturas e epistemologias do sul: as relações internacionais traduzidas pelos movimentos sociais da América Latina

Desde perspectivas poscoloniales, se pretende comprender el tema de la mirada latinoamericana sobre las Relaciones Internacionales, teniendo como ejes las formaciones de identidades de la "periferia" del sistema internacional, las cuales poseen reminiscencias de los procesos coloniales. Por lo tanto, iniciamos con las siguientes preguntas: "¿Cuál es el papel de la cultura en la política mundial?", "Es posible entender las Relaciones Internacionales como un campo de conocimiento sin tener en cuenta las voces silenciadas por los procesos históricos de colonización?", "¿Cuáles son las características del sistema internacional actual y cuál es la relación entre ellas y los procesos locales de subordinación en América Latina? ". En base a estas preguntas, tratamos de contextualizar la disciplina de RI como poseedora de dinámicas de poder internas, que son influenciadas por la coyuntura jerárquica y colonial del sistema internacional, configurando, así, un círculo de prácticas y fundamentos teóricos que afectan localidades fuera de los grandes centros de poder.

Sub-MICs of Mentha piperita essential oil and menthol inhibits AHL mediated quorum sensing and biofilm of Gram-negative bacteria

Bacterial quorum sensing (QS) is a density dependent communication system that regulates the expression of certain genes including production of virulence factors in many pathogens. Bioactive plant extract/compounds inhibiting QS regulated gene expression may be a potential candidate as antipathogenic drug. In this study anti-QS activity of peppermint (Menthe piperita) oil was first tested using the Chromobacterium violaceum CVO26 biosensor. Further, the findings of the present investigation revealed that peppermint oil (PMO) at sub-Minimum Inhibitory Concentrations (sub-MICs) strongly interfered with acyl homoserine lactone (AHL) regulated virulence factors and biofilm formation in Pseudomonas aeruginosa and Aeromonas hydrophila. The result of molecular docking analysis attributed the QS inhibitory activity exhibited by PMO to menthol. Assessment of ability of menthol to interfere with QS systems of various Gram-negative pathogens comprising diverse AHL molecules revealed that it reduced the AHL dependent production of violacein, virulence factors, and biofilm formation indicating broad-spectrum anti-QS activity. Using two Escherichia colt biosensors, MG4/pKDT17 and pEAL08-2, we also confirmed that menthol inhibited both the las and pqs QS systems. Further, findings of the in vivo studies with menthol on nematode model Caenorhabditis elegans showed significantly enhanced survival of the nematode. Our data identified menthol as a novel broad spectrum QS inhibitor.

As relações de trabalho e a resistência escrava em Sant'Ana de Paranaíba: província de Mato Grosso (1828- 1888)

Pós-graduação em História - FCLAS