Evidence of an essential lysine in pig liver 5-aminolevulinic acid dehydratase

5-Aminolevulinic acid dehydratase catalyses the self condensation between two molecules of 5-aminolevulinic acid, via a Schiff base, in which a lysine residue at its active site is proposed to be involved. The aim of the work was to further clarify the mechanism of this step in porphobilinogen biosy...

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Autores principales:	Sopena de Kracoff, Yolanda Elvira, Sancovich, Horacio Alberto
Publicado:	1995
Materias:	5-Aminolevulinic acid dehydratase Active site residues inhibition Glucose effect Lysine residues Pyridoxal 5-phosphate porphobilinogen synthase amino acid sequence animal tissue article controlled study enzyme active site enzyme analysis enzyme purification liver nonhuman swine Animal Enzyme Inhibitors Kinetics Levulinic Acids Liver Lysine Models, Molecular Porphobilinogen Porphobilinogen Synthase Pyridoxal Phosphate Pyridoxamine Pyruvates Pyruvic Acid Support, Non-U.S. Gov't Swine
Acceso en línea:	https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_13572725_v27_n12_p1331_SopenaDeKracoff http://hdl.handle.net/20.500.12110/paper_13572725_v27_n12_p1331_SopenaDeKracoff
Aporte de:	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires

id	paper:paper_13572725_v27_n12_p1331_SopenaDeKracoff
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spelling	paper:paper_13572725_v27_n12_p1331_SopenaDeKracoff2023-06-08T16:11:13Z Evidence of an essential lysine in pig liver 5-aminolevulinic acid dehydratase Sopena de Kracoff, Yolanda Elvira Sancovich, Horacio Alberto 5-Aminolevulinic acid dehydratase Active site residues inhibition Glucose effect Lysine residues Pyridoxal 5-phosphate porphobilinogen synthase amino acid sequence animal tissue article controlled study enzyme active site enzyme analysis enzyme purification liver nonhuman swine Animal Enzyme Inhibitors Kinetics Levulinic Acids Liver Lysine Models, Molecular Porphobilinogen Porphobilinogen Synthase Pyridoxal Phosphate Pyridoxamine Pyruvates Pyruvic Acid Support, Non-U.S. Gov't Swine 5-Aminolevulinic acid dehydratase catalyses the self condensation between two molecules of 5-aminolevulinic acid, via a Schiff base, in which a lysine residue at its active site is proposed to be involved. The aim of the work was to further clarify the mechanism of this step in porphobilinogen biosynthesis. 5-Aminolevulinic acid dehydratase was purified 230-fold from pig liver, ε-Aminolysil residues were identified by treating the enzyme with pyridoxal 5-phosphate. Schiff bases formed between either the substrate or pyridoxal 5-phosphate and the enzyme were stabilized by NaBH4 reduction. Levulinate and pyruvate acted as competitive enzyme inhibitors. Pyridoxal 5-phosphate but not pyridoxamine 5-phosphate reversibly inhibited the enzyme activity, in a competitive fashion (Ki = 0.12 mM). After NaBH4 treatment this inhibition became irreversible. The amount of labelled substrate bound to the enzyme after NaBH4 reduction decreased in the presence of either pyridoxal 5-phosphate, levulinate or pyruvate. Enzyme elution profiles from Sephacryl S-300 showed that NaBH4 treatment (1) in absence of substrate, did not induce any change on the enzyme, eluting as a typical 5-aminolevulinic acid dehydratase single peak (Mw 280,000), which overlapped with that of the enzyme activity; and (2) in the presence of labelled 5-aminolevulinate, had an additional peak eluting with a Mw of 140,000, without enzyme activity. This peak coincides in shape and elution volume with the radioactive one. These data suggest that pyruvate regulates pig liver 5-aminolevulinic acid dehydratase activity in vivo and that during catalysis, a tetrameric structure forms the enzyme-substrate complex. The results support the involvement of a critical ε-aminolysil group at the active site of the enzyme. © 1995 Elsevier Science Ltd. Fil:Sopena De Kracoff, Y.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Sancovich, H.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 1995 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_13572725_v27_n12_p1331_SopenaDeKracoff http://hdl.handle.net/20.500.12110/paper_13572725_v27_n12_p1331_SopenaDeKracoff
institution	Universidad de Buenos Aires
institution_str	I-28
repository_str	R-134
collection	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
topic	5-Aminolevulinic acid dehydratase Active site residues inhibition Glucose effect Lysine residues Pyridoxal 5-phosphate porphobilinogen synthase amino acid sequence animal tissue article controlled study enzyme active site enzyme analysis enzyme purification liver nonhuman swine Animal Enzyme Inhibitors Kinetics Levulinic Acids Liver Lysine Models, Molecular Porphobilinogen Porphobilinogen Synthase Pyridoxal Phosphate Pyridoxamine Pyruvates Pyruvic Acid Support, Non-U.S. Gov't Swine
spellingShingle	5-Aminolevulinic acid dehydratase Active site residues inhibition Glucose effect Lysine residues Pyridoxal 5-phosphate porphobilinogen synthase amino acid sequence animal tissue article controlled study enzyme active site enzyme analysis enzyme purification liver nonhuman swine Animal Enzyme Inhibitors Kinetics Levulinic Acids Liver Lysine Models, Molecular Porphobilinogen Porphobilinogen Synthase Pyridoxal Phosphate Pyridoxamine Pyruvates Pyruvic Acid Support, Non-U.S. Gov't Swine Sopena de Kracoff, Yolanda Elvira Sancovich, Horacio Alberto Evidence of an essential lysine in pig liver 5-aminolevulinic acid dehydratase
topic_facet	5-Aminolevulinic acid dehydratase Active site residues inhibition Glucose effect Lysine residues Pyridoxal 5-phosphate porphobilinogen synthase amino acid sequence animal tissue article controlled study enzyme active site enzyme analysis enzyme purification liver nonhuman swine Animal Enzyme Inhibitors Kinetics Levulinic Acids Liver Lysine Models, Molecular Porphobilinogen Porphobilinogen Synthase Pyridoxal Phosphate Pyridoxamine Pyruvates Pyruvic Acid Support, Non-U.S. Gov't Swine
description	5-Aminolevulinic acid dehydratase catalyses the self condensation between two molecules of 5-aminolevulinic acid, via a Schiff base, in which a lysine residue at its active site is proposed to be involved. The aim of the work was to further clarify the mechanism of this step in porphobilinogen biosynthesis. 5-Aminolevulinic acid dehydratase was purified 230-fold from pig liver, ε-Aminolysil residues were identified by treating the enzyme with pyridoxal 5-phosphate. Schiff bases formed between either the substrate or pyridoxal 5-phosphate and the enzyme were stabilized by NaBH4 reduction. Levulinate and pyruvate acted as competitive enzyme inhibitors. Pyridoxal 5-phosphate but not pyridoxamine 5-phosphate reversibly inhibited the enzyme activity, in a competitive fashion (Ki = 0.12 mM). After NaBH4 treatment this inhibition became irreversible. The amount of labelled substrate bound to the enzyme after NaBH4 reduction decreased in the presence of either pyridoxal 5-phosphate, levulinate or pyruvate. Enzyme elution profiles from Sephacryl S-300 showed that NaBH4 treatment (1) in absence of substrate, did not induce any change on the enzyme, eluting as a typical 5-aminolevulinic acid dehydratase single peak (Mw 280,000), which overlapped with that of the enzyme activity; and (2) in the presence of labelled 5-aminolevulinate, had an additional peak eluting with a Mw of 140,000, without enzyme activity. This peak coincides in shape and elution volume with the radioactive one. These data suggest that pyruvate regulates pig liver 5-aminolevulinic acid dehydratase activity in vivo and that during catalysis, a tetrameric structure forms the enzyme-substrate complex. The results support the involvement of a critical ε-aminolysil group at the active site of the enzyme. © 1995 Elsevier Science Ltd.
author	Sopena de Kracoff, Yolanda Elvira Sancovich, Horacio Alberto
author_facet	Sopena de Kracoff, Yolanda Elvira Sancovich, Horacio Alberto
author_sort	Sopena de Kracoff, Yolanda Elvira
title	Evidence of an essential lysine in pig liver 5-aminolevulinic acid dehydratase
title_short	Evidence of an essential lysine in pig liver 5-aminolevulinic acid dehydratase
title_full	Evidence of an essential lysine in pig liver 5-aminolevulinic acid dehydratase
title_fullStr	Evidence of an essential lysine in pig liver 5-aminolevulinic acid dehydratase
title_full_unstemmed	Evidence of an essential lysine in pig liver 5-aminolevulinic acid dehydratase
title_sort	evidence of an essential lysine in pig liver 5-aminolevulinic acid dehydratase
publishDate	1995
url	https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_13572725_v27_n12_p1331_SopenaDeKracoff http://hdl.handle.net/20.500.12110/paper_13572725_v27_n12_p1331_SopenaDeKracoff
work_keys_str_mv	AT sopenadekracoffyolandaelvira evidenceofanessentiallysineinpigliver5aminolevulinicaciddehydratase AT sancovichhoracioalberto evidenceofanessentiallysineinpigliver5aminolevulinicaciddehydratase
_version_	1768544833375305728

Evidence of an essential lysine in pig liver 5-aminolevulinic acid dehydratase

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