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Previous issue date: 2016-07-29 / A enzima Delta Aminolevul?nico Desidratase (ALAD) ? uma metaloprote?na citos?lica
essencial em v?rios processos biol?gicos, uma vez que ? respons?vel pelo segundo
passo da cat?lise enzim?tica na forma??o de porfobilinog?nio, um precursor dos tetrapirr?licos
(heme, clorofila). Esta enzima ? bastante sens?vel a metais pesados e tem
sido classicamente usada como um marcador na intoxica??o por chumbo. Sua inibi??o
se d? pela substitui??o desses metais pesados no s?tio de liga??o a metais. Na
ALAD humana, o Zinco (Zn2+) ocupa funcionalmente este s?tio sendo essencial para
a coordena??o das cadeias de ?cido aminolevul?nico durante a cat?lise enzim?tica.
Embora muitos ensaios in vitro, in vivo e in s?lico j? tenham demonstrado a import?ncia
do Zn2+ nesse s?tio, n?o se tinha conhecimento de nenhum estudo baseado em
abordagem qu?ntica com o intuito de elucidar esta intera??o de forma mais detalhada.
Diante disso, o presente trabalho teve como objetivo analisar as muta??es missense
que acometem o s?tio de liga??o ao zinco e descrever atrav?s de m?todos qu?nticos a
energia de intera??o entre a enzima e o zinco com maior acur?cia utilizando o m?todo
do Fracionamento Molecular com Capas Conjugadas (MFCC), quantificando energeticamente
os res?duos de amino?cidos posicionados at? uma dist?ncia de 8,5 ? do
centroide do ligante. Foi identificado as altera??es bioqu?micas na estrutura monom?rica
dos mutantes, as quais resultam na diminui??o da atividade enzim?tica. Foram
identificados um total de 30 res?duos com valores energ?ticos variados que interagem
com o zinco no bols?o de liga??o. Aqueles que apresentaram valores significativos
(de atra??o ou repuls?o) e est?o relacionados funcionalmente ? atividade enzim?tica
foram: Lis199, Lis252, Arg 209, Arg 174, Cis122, Cis124 e Cis132; e aqueles que demonstraram
relev?ncia para a perman?ncia do ?on no s?tio de liga??o foram: Asp169,
Gli130, Gli133, Asp120 e Ser168. A partir disso, p?de-se concluir que al?m dos grupos
nucle?filos (grupos tiolatos) dos res?duos Cis122, Cis124 e Cis132, os res?duos
Asp169, Asp120 e Ser168 s?o fundamentais na composi??o do bols?o, uma vez que
demonstraram grande quantidade de energia de intera??o atrativa com o ?on Zn2+. / The enzyme Delta Aminolevulinic Dehydratase (ALAD) is a cytosolic metalloproteinase
essential in several biological processes since it participates in the second step in
porphobilinogen formation pathway, a tetrapyrrolic precursor of heme and chlorophyll.
This enzyme is very sensitive to heavy metals and has traditionally been used as a biomarker
in lead poisoning. Its inhibition occurs when these heavy metals are replaced
inside the metal binding site. In human ALAD, Zinc (Zn2+) functionally occupies this site
and it is essential for coordination of two chains of aminolevulinic acid for the enzymatic
catalysis. Although many in vitro, in vivo and in silico works have already demonstrated
the importance of Zn2+ at that site, to the best of our knowledge, there isn?t any studies
on literature based on quantum approach in order to elucidate this interactions in more
details. Therefore, the aim of the present study was to analyse the missense mutations
that affect the zinc binding site and describe through quantum methods the energy interaction
between zinc and ALAD with greater accuracy using the method of Molecular
fractionation with conjugated caps (MFCC) by quantifying amino acid residues? energy
positioned at 8.5 ? of distance with the ligand centroid. It was identified biochemical
changes in the monomeric structure of mutants, which result in decreased enzyme
activity. It were identified a total of 30 residues with a wide range of energy values.
The residues with significant (atractition or repulsion) values and functionally related to
enzymatic activity were: Lys199, Lys252, Cys122, Cys124 and Cys132; and those that
demonstrated relevance to the ion permanence inside the binding site were: Asp169,
Gly130, Gly133, Asp120 and Ser168. Thus, it could be concluded that in addition to
the nucleophilic groups (thiolates groups) from Cys122, Cys124 and Cys132, others
residues such as Asp169, Asp120 and Ser168 are fundamental in the catalytic pocket
composition, since they showed high attractive interaction energy with Zn2+ ion.
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/21908 |
| Date | 29 July 2016 |
| Creators | Barbosa, Emmanuel Duarte |
| Contributors | 91206359072, Fulco, Umberto Laino, 67196675487, Sinigaglia, Marialva, 60901691020, Amaral, Viviane Souza do |
| Publisher | PROGRAMA DE P?S-GRADUA??O EM BIOQU?MICA, UFRN, Brasil |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Source | reponame:Repositório Institucional da UFRN, instname:Universidade Federal do Rio Grande do Norte, instacron:UFRN |
| Rights | info:eu-repo/semantics/openAccess |
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