Rat kidney porphobilinogen deaminase kinetics: Detection of enzyme-substrate complexes

Background and aims: Acute intermittent porphyria (AIP) is an inherited disease resulting from a reduced activity of the enzyme porphobilinogen deaminase (PBG-D). The kidney is an important target for numerous porphyrinogenic drugs and it may contribute to the clinical manifestations of porphyric at...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Noriega, Guillermo Osvaldo, Batlle, Alcira María del Carmen, Juknat, Adela Ana
Publicado:	2002
Materias:	Enzyme-substrate complexes Isoenzymes Kinetic mechanism Porphobilinogen deaminase Rat kidney porphobilinogen deaminase isoenzyme kidney enzyme uroporphyrinogen animal article enzyme specificity enzymology isoelectric focusing kidney kinetics male metabolism polyacrylamide gel electrophoresis rat Western blotting animal tissue conformational transition controlled study enzyme active site enzyme purification enzyme substrate complex incubation time molecular cloning molecular weight nonhuman pH protein expression Animals Blotting, Western Electrophoresis, Polyacrylamide Gel Hydroxymethylbilane Synthase Isoelectric Focusing Kidney Kinetics Male Rats Substrate Specificity Animal Support, Non-U.S. Gov't
Acceso en línea:	https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_13572725_v34_n10_p1230_Noriega http://hdl.handle.net/20.500.12110/paper_13572725_v34_n10_p1230_Noriega
Aporte de:	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires

id	paper:paper_13572725_v34_n10_p1230_Noriega
record_format	dspace
spelling	paper:paper_13572725_v34_n10_p1230_Noriega2023-06-08T16:11:17Z Rat kidney porphobilinogen deaminase kinetics: Detection of enzyme-substrate complexes Noriega, Guillermo Osvaldo Batlle, Alcira María del Carmen Juknat, Adela Ana Enzyme-substrate complexes Isoenzymes Kinetic mechanism Porphobilinogen deaminase Rat kidney porphobilinogen deaminase isoenzyme kidney enzyme porphobilinogen deaminase uroporphyrinogen animal article enzyme specificity enzymology isoelectric focusing kidney kinetics male metabolism polyacrylamide gel electrophoresis rat Western blotting animal tissue conformational transition controlled study enzyme active site enzyme purification enzyme substrate complex incubation time kidney molecular cloning molecular weight nonhuman pH protein expression Animals Blotting, Western Electrophoresis, Polyacrylamide Gel Hydroxymethylbilane Synthase Isoelectric Focusing Kidney Kinetics Male Rats Substrate Specificity Animal Blotting, Western Electrophoresis, Polyacrylamide Gel Isoelectric Focusing Kinetics Male Rats Substrate Specificity Support, Non-U.S. Gov't Background and aims: Acute intermittent porphyria (AIP) is an inherited disease resulting from a reduced activity of the enzyme porphobilinogen deaminase (PBG-D). The kidney is an important target for numerous porphyrinogenic drugs and it may contribute to the clinical manifestations of porphyric attacks. An evaluation of kidney PBG-D role in the AIP pathophysiology requires detailed information on kidney PBG-D properties, under normal conditions. Methods: Rat kidney PBG-D was purified to homogeneity and initial reaction velocities were calculated by measuring uroporphyrinogen I formation at pH 8.2 for different incubation times (0-20min) and over a wide range of substrate concentrations (0.8-66μM). Results: Purified rat kidney PBG-D is a monomeric enzyme showing only a single protein band after SDS-PAGE, Western blot and isoelectric focusing (pI 4.9). Its molecular mass is 40±2.3kDa, determined by SDS-PAGE and 39.8±2kDa by gel filtration chromatography. Rat kidney PBG-D has an unusual kinetic behaviour, exhibiting a deviation from the Michaelis-Menten hyperbola. PBG-D kinetic data required a fitting to an equation of higher degree, leading to the following apparent kinetic constants: K1=2.08±0.01μM and K2=0.102±0.003μM. Conclusion: The values of these constants fulfil the restriction 4K2≤K1 2, necessary for the occurrence of isoenzymes, interpreted in this work as enzyme-substrate intermediates. The initial reaction velocity expression here defined, correlates with an enzyme carrying only one active site but allowing, through conformational changes, the detection of at least two enzyme-substrate intermediates formed during PBG-D reaction. © 2002 Elsevier Science Ltd. All rights reserved. Fil:Noriega, G. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Batlle, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Juknat, A.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 2002 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_13572725_v34_n10_p1230_Noriega http://hdl.handle.net/20.500.12110/paper_13572725_v34_n10_p1230_Noriega
institution	Universidad de Buenos Aires
institution_str	I-28
repository_str	R-134
collection	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
topic	Enzyme-substrate complexes Isoenzymes Kinetic mechanism Porphobilinogen deaminase Rat kidney porphobilinogen deaminase isoenzyme kidney enzyme porphobilinogen deaminase uroporphyrinogen animal article enzyme specificity enzymology isoelectric focusing kidney kinetics male metabolism polyacrylamide gel electrophoresis rat Western blotting animal tissue conformational transition controlled study enzyme active site enzyme purification enzyme substrate complex incubation time kidney molecular cloning molecular weight nonhuman pH protein expression Animals Blotting, Western Electrophoresis, Polyacrylamide Gel Hydroxymethylbilane Synthase Isoelectric Focusing Kidney Kinetics Male Rats Substrate Specificity Animal Blotting, Western Electrophoresis, Polyacrylamide Gel Isoelectric Focusing Kinetics Male Rats Substrate Specificity Support, Non-U.S. Gov't
spellingShingle	Enzyme-substrate complexes Isoenzymes Kinetic mechanism Porphobilinogen deaminase Rat kidney porphobilinogen deaminase isoenzyme kidney enzyme porphobilinogen deaminase uroporphyrinogen animal article enzyme specificity enzymology isoelectric focusing kidney kinetics male metabolism polyacrylamide gel electrophoresis rat Western blotting animal tissue conformational transition controlled study enzyme active site enzyme purification enzyme substrate complex incubation time kidney molecular cloning molecular weight nonhuman pH protein expression Animals Blotting, Western Electrophoresis, Polyacrylamide Gel Hydroxymethylbilane Synthase Isoelectric Focusing Kidney Kinetics Male Rats Substrate Specificity Animal Blotting, Western Electrophoresis, Polyacrylamide Gel Isoelectric Focusing Kinetics Male Rats Substrate Specificity Support, Non-U.S. Gov't Noriega, Guillermo Osvaldo Batlle, Alcira María del Carmen Juknat, Adela Ana Rat kidney porphobilinogen deaminase kinetics: Detection of enzyme-substrate complexes
topic_facet	Enzyme-substrate complexes Isoenzymes Kinetic mechanism Porphobilinogen deaminase Rat kidney porphobilinogen deaminase isoenzyme kidney enzyme porphobilinogen deaminase uroporphyrinogen animal article enzyme specificity enzymology isoelectric focusing kidney kinetics male metabolism polyacrylamide gel electrophoresis rat Western blotting animal tissue conformational transition controlled study enzyme active site enzyme purification enzyme substrate complex incubation time kidney molecular cloning molecular weight nonhuman pH protein expression Animals Blotting, Western Electrophoresis, Polyacrylamide Gel Hydroxymethylbilane Synthase Isoelectric Focusing Kidney Kinetics Male Rats Substrate Specificity Animal Blotting, Western Electrophoresis, Polyacrylamide Gel Isoelectric Focusing Kinetics Male Rats Substrate Specificity Support, Non-U.S. Gov't
description	Background and aims: Acute intermittent porphyria (AIP) is an inherited disease resulting from a reduced activity of the enzyme porphobilinogen deaminase (PBG-D). The kidney is an important target for numerous porphyrinogenic drugs and it may contribute to the clinical manifestations of porphyric attacks. An evaluation of kidney PBG-D role in the AIP pathophysiology requires detailed information on kidney PBG-D properties, under normal conditions. Methods: Rat kidney PBG-D was purified to homogeneity and initial reaction velocities were calculated by measuring uroporphyrinogen I formation at pH 8.2 for different incubation times (0-20min) and over a wide range of substrate concentrations (0.8-66μM). Results: Purified rat kidney PBG-D is a monomeric enzyme showing only a single protein band after SDS-PAGE, Western blot and isoelectric focusing (pI 4.9). Its molecular mass is 40±2.3kDa, determined by SDS-PAGE and 39.8±2kDa by gel filtration chromatography. Rat kidney PBG-D has an unusual kinetic behaviour, exhibiting a deviation from the Michaelis-Menten hyperbola. PBG-D kinetic data required a fitting to an equation of higher degree, leading to the following apparent kinetic constants: K1=2.08±0.01μM and K2=0.102±0.003μM. Conclusion: The values of these constants fulfil the restriction 4K2≤K1 2, necessary for the occurrence of isoenzymes, interpreted in this work as enzyme-substrate intermediates. The initial reaction velocity expression here defined, correlates with an enzyme carrying only one active site but allowing, through conformational changes, the detection of at least two enzyme-substrate intermediates formed during PBG-D reaction. © 2002 Elsevier Science Ltd. All rights reserved.
author	Noriega, Guillermo Osvaldo Batlle, Alcira María del Carmen Juknat, Adela Ana
author_facet	Noriega, Guillermo Osvaldo Batlle, Alcira María del Carmen Juknat, Adela Ana
author_sort	Noriega, Guillermo Osvaldo
title	Rat kidney porphobilinogen deaminase kinetics: Detection of enzyme-substrate complexes
title_short	Rat kidney porphobilinogen deaminase kinetics: Detection of enzyme-substrate complexes
title_full	Rat kidney porphobilinogen deaminase kinetics: Detection of enzyme-substrate complexes
title_fullStr	Rat kidney porphobilinogen deaminase kinetics: Detection of enzyme-substrate complexes
title_full_unstemmed	Rat kidney porphobilinogen deaminase kinetics: Detection of enzyme-substrate complexes
title_sort	rat kidney porphobilinogen deaminase kinetics: detection of enzyme-substrate complexes
publishDate	2002
url	https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_13572725_v34_n10_p1230_Noriega http://hdl.handle.net/20.500.12110/paper_13572725_v34_n10_p1230_Noriega
work_keys_str_mv	AT noriegaguillermoosvaldo ratkidneyporphobilinogendeaminasekineticsdetectionofenzymesubstratecomplexes AT batllealciramariadelcarmen ratkidneyporphobilinogendeaminasekineticsdetectionofenzymesubstratecomplexes AT juknatadelaana ratkidneyporphobilinogendeaminasekineticsdetectionofenzymesubstratecomplexes
_version_	1768543049794715648

Rat kidney porphobilinogen deaminase kinetics: Detection of enzyme-substrate complexes

Ejemplares similares