Mechanism of activation of camp-dependent protein kinase: In mucor rouxii the apparent specific activity of the camp-activated holoenzyme is different than that of its free catalytic subunit

Kinetic constants for peptide phosphorylation by the catalytic subunit of the dimorphic fungus Mucor rouxii protein kinase A were determined using 13 peptides derived from the peptide containing the basic consensus sequence RRASVA, plus kemptide, S6 peptide, and protamine. As a whole, although with...

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Autores principales:	Zaremberg, Vanina, Moreno de Colonna, Silvia
Publicado:	2000
Materias:	CAMP activation Catalytic subunit Mucor rouxii Protein kinase A Substrate cyclic AMP dependent protein kinase polycation animal cell article binding affinity catalysis concentration response controlled study enzyme activation enzyme activity enzyme inhibition kinetics nonhuman phosphorylation priority journal
Acceso en línea:	https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00039861_v381_n1_p74_Zaremberg http://hdl.handle.net/20.500.12110/paper_00039861_v381_n1_p74_Zaremberg
Aporte de:	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires

id	paper:paper_00039861_v381_n1_p74_Zaremberg
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spelling	paper:paper_00039861_v381_n1_p74_Zaremberg2023-06-08T14:25:03Z Mechanism of activation of camp-dependent protein kinase: In mucor rouxii the apparent specific activity of the camp-activated holoenzyme is different than that of its free catalytic subunit Zaremberg, Vanina Moreno de Colonna, Silvia CAMP activation Catalytic subunit Mucor rouxii Protein kinase A Substrate cyclic AMP dependent protein kinase polycation animal cell article binding affinity catalysis concentration response controlled study enzyme activation enzyme activity enzyme inhibition kinetics nonhuman phosphorylation priority journal Kinetic constants for peptide phosphorylation by the catalytic subunit of the dimorphic fungus Mucor rouxii protein kinase A were determined using 13 peptides derived from the peptide containing the basic consensus sequence RRASVA, plus kemptide, S6 peptide, and protamine. As a whole, although with a greater K(m), the order of preference of the peptides by the M. rouxii catalytic subunit was similar to the one displayed by mammalian protein kinase A. Particularly significant is the replacement of serine by threonine in the basic peptide RRATVA, which impaired its role as a substrate of M. rouxii catalytic subunit. Mucor rouxii protein kinase A is a good model in which to study the mechanism of activation since cAMP alone is not enough to promote activation and dissociation. Four peptides were selected for the study of holoenzyme activation under conditions in which the enzymatic activity was not proportional to the holoenzyme concentration: RRASVA, RRRRASVA, KRRRLSSRA (S6 peptide), and LRRASLG (kemptide); protamine was used as reference. Differential activation degree was observed depending on the peptide used and on cAMP concentration. Ratios of activity between different substrates displayed by the holoenzyme under the above conditions did not reflect the one expected for the free catalytic subunit. The degree of inhibition of the holoenzyme activity by an active peptide derived from the thermostable protein kinase inhibitor was dependent on the substrate used and on the holoenzyme concentration, while it was found to be independent of these two parameters for free catalytic subunit. Polycation modulation of holoenzyme activation by cAMP was also dependent on the polycation itself and on the peptide used as substrate. The observed kinetic differences between holoenzyme and free catalytic subunit were decreased or almost abolished when working at low enzyme or at high cAMP concentrations. Two hypotheses compatible with the results are discussed: Substrate participation in the dissociation process and/or holoenzyme activation without dissociation. (C) 2000 Academic Press. Fil:Zaremberg, V. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Moreno, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 2000 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00039861_v381_n1_p74_Zaremberg http://hdl.handle.net/20.500.12110/paper_00039861_v381_n1_p74_Zaremberg
institution	Universidad de Buenos Aires
institution_str	I-28
repository_str	R-134
collection	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
topic	CAMP activation Catalytic subunit Mucor rouxii Protein kinase A Substrate cyclic AMP dependent protein kinase polycation animal cell article binding affinity catalysis concentration response controlled study enzyme activation enzyme activity enzyme inhibition kinetics nonhuman phosphorylation priority journal
spellingShingle	CAMP activation Catalytic subunit Mucor rouxii Protein kinase A Substrate cyclic AMP dependent protein kinase polycation animal cell article binding affinity catalysis concentration response controlled study enzyme activation enzyme activity enzyme inhibition kinetics nonhuman phosphorylation priority journal Zaremberg, Vanina Moreno de Colonna, Silvia Mechanism of activation of camp-dependent protein kinase: In mucor rouxii the apparent specific activity of the camp-activated holoenzyme is different than that of its free catalytic subunit
topic_facet	CAMP activation Catalytic subunit Mucor rouxii Protein kinase A Substrate cyclic AMP dependent protein kinase polycation animal cell article binding affinity catalysis concentration response controlled study enzyme activation enzyme activity enzyme inhibition kinetics nonhuman phosphorylation priority journal
description	Kinetic constants for peptide phosphorylation by the catalytic subunit of the dimorphic fungus Mucor rouxii protein kinase A were determined using 13 peptides derived from the peptide containing the basic consensus sequence RRASVA, plus kemptide, S6 peptide, and protamine. As a whole, although with a greater K(m), the order of preference of the peptides by the M. rouxii catalytic subunit was similar to the one displayed by mammalian protein kinase A. Particularly significant is the replacement of serine by threonine in the basic peptide RRATVA, which impaired its role as a substrate of M. rouxii catalytic subunit. Mucor rouxii protein kinase A is a good model in which to study the mechanism of activation since cAMP alone is not enough to promote activation and dissociation. Four peptides were selected for the study of holoenzyme activation under conditions in which the enzymatic activity was not proportional to the holoenzyme concentration: RRASVA, RRRRASVA, KRRRLSSRA (S6 peptide), and LRRASLG (kemptide); protamine was used as reference. Differential activation degree was observed depending on the peptide used and on cAMP concentration. Ratios of activity between different substrates displayed by the holoenzyme under the above conditions did not reflect the one expected for the free catalytic subunit. The degree of inhibition of the holoenzyme activity by an active peptide derived from the thermostable protein kinase inhibitor was dependent on the substrate used and on the holoenzyme concentration, while it was found to be independent of these two parameters for free catalytic subunit. Polycation modulation of holoenzyme activation by cAMP was also dependent on the polycation itself and on the peptide used as substrate. The observed kinetic differences between holoenzyme and free catalytic subunit were decreased or almost abolished when working at low enzyme or at high cAMP concentrations. Two hypotheses compatible with the results are discussed: Substrate participation in the dissociation process and/or holoenzyme activation without dissociation. (C) 2000 Academic Press.
author	Zaremberg, Vanina Moreno de Colonna, Silvia
author_facet	Zaremberg, Vanina Moreno de Colonna, Silvia
author_sort	Zaremberg, Vanina
title	Mechanism of activation of camp-dependent protein kinase: In mucor rouxii the apparent specific activity of the camp-activated holoenzyme is different than that of its free catalytic subunit
title_short	Mechanism of activation of camp-dependent protein kinase: In mucor rouxii the apparent specific activity of the camp-activated holoenzyme is different than that of its free catalytic subunit
title_full	Mechanism of activation of camp-dependent protein kinase: In mucor rouxii the apparent specific activity of the camp-activated holoenzyme is different than that of its free catalytic subunit
title_fullStr	Mechanism of activation of camp-dependent protein kinase: In mucor rouxii the apparent specific activity of the camp-activated holoenzyme is different than that of its free catalytic subunit
title_full_unstemmed	Mechanism of activation of camp-dependent protein kinase: In mucor rouxii the apparent specific activity of the camp-activated holoenzyme is different than that of its free catalytic subunit
title_sort	mechanism of activation of camp-dependent protein kinase: in mucor rouxii the apparent specific activity of the camp-activated holoenzyme is different than that of its free catalytic subunit
publishDate	2000
url	https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00039861_v381_n1_p74_Zaremberg http://hdl.handle.net/20.500.12110/paper_00039861_v381_n1_p74_Zaremberg
work_keys_str_mv	AT zarembergvanina mechanismofactivationofcampdependentproteinkinaseinmucorrouxiitheapparentspecificactivityofthecampactivatedholoenzymeisdifferentthanthatofitsfreecatalyticsubunit AT morenodecolonnasilvia mechanismofactivationofcampdependentproteinkinaseinmucorrouxiitheapparentspecificactivityofthecampactivatedholoenzymeisdifferentthanthatofitsfreecatalyticsubunit
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Mechanism of activation of camp-dependent protein kinase: In mucor rouxii the apparent specific activity of the camp-activated holoenzyme is different than that of its free catalytic subunit

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