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...
Guardado en:
| Autores principales: | , , , |
|---|---|
| Formato: | JOUR |
| Materias: | |
| Acceso en línea: | http://hdl.handle.net/20.500.12110/paper_13572725_v34_n10_p1230_Noriega |
| Aporte de: |
| id |
todo:paper_13572725_v34_n10_p1230_Noriega |
|---|---|
| record_format |
dspace |
| spelling |
todo:paper_13572725_v34_n10_p1230_Noriega2023-10-03T16:10:29Z Rat kidney porphobilinogen deaminase kinetics: Detection of enzyme-substrate complexes Noriega, G. Mattei, G. Batlle, A. Juknat, A.A. 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. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar 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, G. Mattei, G. Batlle, A. Juknat, A.A. 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. |
| format |
JOUR |
| author |
Noriega, G. Mattei, G. Batlle, A. Juknat, A.A. |
| author_facet |
Noriega, G. Mattei, G. Batlle, A. Juknat, A.A. |
| author_sort |
Noriega, G. |
| 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 |
| url |
http://hdl.handle.net/20.500.12110/paper_13572725_v34_n10_p1230_Noriega |
| work_keys_str_mv |
AT noriegag ratkidneyporphobilinogendeaminasekineticsdetectionofenzymesubstratecomplexes AT matteig ratkidneyporphobilinogendeaminasekineticsdetectionofenzymesubstratecomplexes AT batllea ratkidneyporphobilinogendeaminasekineticsdetectionofenzymesubstratecomplexes AT juknataa ratkidneyporphobilinogendeaminasekineticsdetectionofenzymesubstratecomplexes |
| _version_ |
1807315602862243840 |