{"id":117956,"date":"2018-03-11T10:47:44","date_gmt":"2018-03-11T10:47:44","guid":{"rendered":"https:\/\/www.deberes.net\/tesis\/sin-categoria\/nuevos-aspectos-en-las-actividades-catala%c2%adticas-de-tirosinasa\/"},"modified":"2018-03-11T10:47:44","modified_gmt":"2018-03-11T10:47:44","slug":"nuevos-aspectos-en-las-actividades-catala%c2%adticas-de-tirosinasa","status":"publish","type":"post","link":"https:\/\/www.deberes.net\/tesis\/bioquimica-molecular\/nuevos-aspectos-en-las-actividades-catala%c2%adticas-de-tirosinasa\/","title":{"rendered":"Nuevos aspectos en las actividades catal\u00edticas de tirosinasa"},"content":{"rendered":"<h2>Tesis doctoral de <strong> Mar\u00eda  Del Mar Garcia Molina <\/strong><\/h2>\n<p>Objetivos: el objetivo fundamental de esta memoria consiste en profundizar en el mecanismo de acci\u00f3n de la tirosinasa. Se centra la atenci\u00f3n de este estudio sobre la actividad monofenolasa y se profundiza en el proceso de inactivaci\u00f3n suicida. Se investiga la aportaci\u00f3n de cada una de las actividades monofenolasa y difenolasa en dicho proceso. Se aborda la caracterizaci\u00f3n cin\u00e9tica de monofenoles de inter\u00e9s fisiol\u00f3gico. Metodolog\u00eda: 1. Profundizar en el mecanismo cin\u00e9tico de actuaci\u00f3n de la enzima en sus actividades monofenolasa y difenolasa. 2. Estudiar la hidroxilaci\u00f3n de umbeliferona a esculetina en la ruta de bios\u00edntesis de cumarinas. 3. Establecer una metodolog\u00eda para investigar si un monofenol es inhibidor o sustrato alternativo de la enzima con la ayuda del per\u00f3xido de hidr\u00f3geno. 4. Diferenciar las actividades monofenolasa y difenolasa en su implicaci\u00f3n en el proceso de inactivaci\u00f3n suicida. 5. Profundizar en el estudio del mecanismo de inactivaci\u00f3n suicida de la enzima en su acci\u00f3n sobre o-difenoles mediante la determinaci\u00f3n del efecto isot\u00f3pico generado en un medio deuterado. 6. Estudiar y analizar los procesos de cat\u00e1lisis e inactivaci\u00f3n en la acci\u00f3n de tirosinasa sobre tres tipos de sustratos: o-difenoles, o-aminofenoles y o-fenilendiaminas. 7. Estudiar la acci\u00f3n de tirosinasa sobre hidroxihidroquinona en los procesos de cat\u00e1lisis e inactivaci\u00f3n. 8. Estudiar la reacci\u00f3n de tirosinasa con hidroquinona, un monofenol, utilizado como despigmentante. 9. Caracterizar cin\u00e9ticamente a hidroquinona como un sustrato de tirosinasa con la ayuda del per\u00f3xido de hidr\u00f3geno. 10. Estudiar la influencia de \u00e1cido asc\u00f3rbico sobre el proceso de hidroxilaci\u00f3n de hidroquinona por tirosinasa. 10. Caracterizar cin\u00e9ticamente a hidroquinona como un sustrato de tirosinasa con la ayuda de cantidades catal\u00edticas de o-difenol y \u00e1cido asc\u00f3rbico. Resultados: 1. Se ha demostrado que tirosinasa podr\u00eda participar en la ruta de bios\u00edntesis de cumarinas hidroxilando umbeliferona a esculetina. 2. Per\u00f3xido de hidr\u00f3geno ayuda a demostrar si un monofenol es sustrato o inhibidor de tirosinasa actuando sobre la forma   (metatirosinasa) y transform\u00e1ndola en la forma   (oxitirosinasa). 3. Los estudios cin\u00e9ticos con tbf\/tbc demuestran que la enzima se inactiva \u00fanicamente al actuar sobre o-difenoles. 4. Un conjunto de estudios de efecto isot\u00f3pico con d2o han puesto de manifiesto que existe una etapa muy lenta en la que se transfiere un prot\u00f3n. Esta etapa podr\u00eda ser previa a la inactivaci\u00f3n suicida. 5. La cat\u00e1lisis de la oxidaci\u00f3n de o-difenoles, o-aminofenoles y o-fenilendiaminas, sigue un mecanismo similar: oxidaci\u00f3n\/ reducci\u00f3n simult\u00e1nea sobre los dos \u00e1tomos de cobre. 6. El proceso de inactivaci\u00f3n suicida en la acci\u00f3n de la enzima sobre estos sustratos parece seguir un mismo mecanismo, que podr\u00eda ser la oxidaci\u00f3n\/ reducci\u00f3n de uno de los \u00e1tomos de cobre. 7. Los efectos electr\u00f3nicos de los sustituyentes de c-4 de o-difenoles, o-aminofenoles y o-fenilendiaminas en las etapas de cat\u00e1lisis e inactivaci\u00f3n son m\u00e1s importantes en los o-difenoles y o-aminofenles. 8. El producto de hidroxilaci\u00f3n de hidroquinona, la hidroxihidroquinona, es un sustrato suicida de la enzima. 9. Se ha puesto de manifiesto que hidroquinona, un agente despigmentante, es un sustrato de tirosinasa. 10. Se ha demostrado que tirosinasa act\u00faa sobre hidroquinona en presencia de ox\u00edgeno molecular y per\u00f3xido de hidr\u00f3geno, ya que este \u00faltimo pasa metatirosinasa (inactiva sobre hidroquinona) a oxitirosinasa (activa sobre hidroquinona). 11. Tambi\u00e9n se ha puesto de manifiesto que tirosinasa act\u00faa sobre hidroquinona en presencia de ox\u00edgeno y \u00e1cido asc\u00f3rbico a altas concentraciones, por el paso de metatirosinasa a desoxitirosinasa. Del mismo modo, cantidades catal\u00edticas de tert-butilcatecol y \u00e1cido asc\u00f3rbico en concentraciones de orden micromolar consiguen la acci\u00f3n de tirosinasa sobre hidroquinona a trav\u00e9s del paso de metatirosinasa a desoxitirosinasa. Abstract objectives: the main objective of this report is to deepen the mechanism of action of tyrosinase. The focus of this study is on the monophenolase activity and deepened in the process of suicide inactivation. The contribution of each of the monophenolase and diphenolase activities in this process is investigated. In this regard, the kinetic characterization of monophenols with physiological interest is studied. 1. Deepen in the kinetic mechanism of tyrosinase in their monophenolase and diphenolase activities. 2. Study the hydroxilation of umbelliferone to scueltin in the cumarin biosynthesis pathway. 3. Establish a methodology to investigate if one monophenol is inhibitor or alternative substrate of tyrosinase with the aid of hydrogen peroxide. 4. Discriminate between the monophenolase and diphenolase activities in its involvement on the suicide inactivation process. 5. Deepen the study of suicide inactivation process of tyrosinase in its action on o-diphenols through the determination of isotopic effect generated in a deuterated medium. 6. Study and analyse the catalysis and inactivation processes in the action of tyrosinase on tree types of substrates: o-diphenols, o-aminophenols and o-phenylendiamines.  7. Study the tyrosinase action on hydroxyhydroquinone in the catalysis and inactivation processes. 8. Investigate the action of tyrosinase on a monophenol, hydroquinone, used as lightening. 9. Kinetically characterize to hydroquinone as a substrate of tyrosinase with the aid of hydrogen peroxide. 10. Study the ascorbic acid influence on the hydroquinone hydroxylation process by tyrosinase. 11. Kinetically characterize to hydroquinone as a substrate of tyrosinase with the help of catalytic amounts of o-diphenol and ascorbic acid. Results: 1. It has been shown that tyrosinase could be involved in the biosynthesis pathway of the coumarins by the hydroxylation of umbelliferone to esculetin. 2. Hydrogen peroxide helps to demonstrate whether a monophenol is a substrate or an inhibitor of tyrosinase acting on the form   (metatyrosinase) and converting it into the form   (oxytyrosinase). 3. The kinetic studies with tbf \/ tbc show that the enzyme is only inactivated when it acts on o-diphenols. 4. A set of studies of the isotopic effect in d2o have shown that there is a very slow step in which a proton is transferred. It could be considered as a previous step of the suicide inactivation. 5. The catalysis of the oxidation of o-diphenols, aminophenols and o-phenylenediamines occurs through a similar mechanism: simultaneous oxidation \/ reduction of the two copper atoms. 6. The process of suicide inactivation in the action of the enzyme on these substrates seems to follow the same mechanism which could suppose the oxidation \/ reduction of one of the copper atoms. 7. The electronic effects of the substituents on the carbon atom c-4 of o-diphenols, o-aminophenols and o-phenylenediamines during the catalytic and inactivation steps are more significant in the case of the o-diphenols and o-aminophenols. 8. The product of the hydroxylation of hydroquinone, hydroxyhydroquinone, is a suicide substrate of the enzyme. 9. It has been shown that hydroquinone, a depigmenting agent, is a substrate of tyrosinase. 10. It has been shown that tyrosinase acts on hydroquinone in the presence of molecular oxygen and hydrogen peroxide because the latter converts metatyrosinase (inactive on hydroquinone) to oxytyrosinase (active on hydroquinone). 11. It has also been found that tyrosinase acts on hydroquinone in the presence of molecular oxygen and ascorbic acid at high concentrations, by transforming metatyrosinase to desoxytyrosinase. Similarly, catalytic amounts of tert-butylcatechol and ascorbic acid at micromolar concentrations induce the action of tyrosinase on hydroquinone by the conversion of metatyrosinase to desoxytyrosinase step.<\/p>\n<p>&nbsp;<\/p>\n<h3>Datos acad\u00e9micos de la tesis doctoral \u00ab<strong>Nuevos aspectos en las actividades catal\u00edticas de tirosinasa<\/strong>\u00ab<\/h3>\n<ul>\n<li><strong>T\u00edtulo de la tesis:<\/strong>\u00a0 Nuevos aspectos en las actividades catal\u00edticas de tirosinasa <\/li>\n<li><strong>Autor:<\/strong>\u00a0 Mar\u00eda  Del Mar Garcia Molina <\/li>\n<li><strong>Universidad:<\/strong>\u00a0 Murcia<\/li>\n<li><strong>Fecha de lectura de la tesis:<\/strong>\u00a0 24\/07\/2015<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n<h3>Direcci\u00f3n y tribunal<\/h3>\n<ul>\n<li><strong>Director de la tesis<\/strong>\n<ul>\n<li>Francisco Garcia Molina<\/li>\n<\/ul>\n<\/li>\n<li><strong>Tribunal<\/strong>\n<ul>\n<li>Presidente del tribunal: Mar\u00eda  llanos Amo saus <\/li>\n<li>milagros Molina alarcon (vocal)<\/li>\n<li>  (vocal)<\/li>\n<li>  (vocal)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Tesis doctoral de Mar\u00eda Del Mar Garcia Molina Objetivos: el objetivo fundamental de esta memoria consiste en profundizar en el [&hellip;]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"site-sidebar-layout":"default","site-content-layout":"","ast-site-content-layout":"","site-content-style":"default","site-sidebar-style":"default","ast-global-header-display":"","ast-banner-title-visibility":"","ast-main-header-display":"","ast-hfb-above-header-display":"","ast-hfb-below-header-display":"","ast-hfb-mobile-header-display":"","site-post-title":"","ast-breadcrumbs-content":"","ast-featured-img":"","footer-sml-layout":"","theme-transparent-header-meta":"","adv-header-id-meta":"","stick-header-meta":"","header-above-stick-meta":"","header-main-stick-meta":"","header-below-stick-meta":"","astra-migrate-meta-layouts":"default","ast-page-background-enabled":"default","ast-page-background-meta":{"desktop":{"background-color":"var(--ast-global-color-4)","background-image":"","background-repeat":"repeat","background-position":"center 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