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Previous issue date: 2008-07-02 === Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior === Serines proteinases inhibitors (PIs) are widely distributed in nature and are able to inhibit both in vitro and in vivo enzymatic activites. Seed PIs in than leguminous are
classified in seven families, Bowman-Birk and Kunitz type families that most studied representing an important role in the first line of defense toward insects pests. Some
Kunitz type inhibitors possess activities serine and cysteine for proteinases named bifunctional inhibitor, as ApTKI the inhibitor isolate from seed of Adenanthera pavonina.
The A. pavonina inhibitor presenting the uncommon property and was used for interaction studies between proteinases serine (trypsin) and cysteine (papain). In order
to determinate the in vitro interaction of ApTKI against enzymes inhibitor purification was carried cut by using chromatographic techniques and inhibition assays. The 3D model of the bifunctional inhibitor ApTKI was constructed SWISS-MODEL program by homology modeling using soybean trypsin inhibitor (STI, pdb:1ba7), as template which presented
40% of identity to A. pavonina inhibitor. Model quality was evaluated by PROCHECK program. Moreover in silico analyzes of formed complex between the enzymes and
ApTKI was evaluated by HEX 4.5 program. In vitro results confirmed the inhibitory assays, where the inhibitor presented the ability to simultaneously inhibit trypsin and
papain. The residues encountered in the inhibitor model of folder structural three-dimensional that make contact to enzymes target coud explain the specificity pattern
against serine and cysteine proteinases === Os inibidores de proteinases ser?nicas (IPs) est?o extensamente distribu?dos na natureza e inibem a atividade enzim?tica in vitro e in vivo. Estes IPs em sementes de
leguminosas compreendem sete fam?lias, no entanto as fam?lias Bowman-Birk e do tipo Kunitz s?o as mais estudadas e representam um papel importante na primeira linha de
defesa contra insetos pragas. Alguns inibidores do tipo Kunitz possuem atividades para proteinases ser?nicas e ciste?nicas sendo denominados inibidores bifuncionais, como o inibidor ApTKI da semente de Adenanthera pavonina. O inibidor de A. pavonina por apresentar essa caracter?stica incomum aos inibidores dessa fam?lia foi utilizado para o estudo da intera??o entre as proteinases ser?nica (tripsina) e ciste?nica (papa?na). Para determinar a intera??o in vitro de ApTKI e as enzimas alvo foi realizada a purifica??o do inibidor a partir de t?cnicas cromatogr?ficas e ensaios de inibi??o. O modelo 3D do
inibidor bifuncional ApTKI foi constru?do pelo programa SWISS-MODEL atrav?s da metodologia de modelagem por homologia utilizando como molde o inibidor de tripsina de soja (STI, pdb:1ba7) que apresentou 40% de identidade com a prote?na alvo. A qualidade do modelo foi avaliada pelo programa PROCHECK. Para a an?lise do complexo in silico entre as enzimas alvo e o inibidor foi utilizado o programa HEX 4.5. Estes resultados confirmaram os ensaios inibit?rios in vitro, onde ApTKI apresentou a capacidade de inibir simultaneamente tripsina e papa?na. Algumas das diferen?as
observadas nos res?duos do sitio reativo explicam a forte afinidade para tripsina e a fraca para papa?na
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