Cargando…

Mixed Disulfide Formation at Cys141 Leads to Apparent Unidirectional Attenuation of Aspergillus niger NADP-Glutamate Dehydrogenase Activity

NADP-Glutamate dehydrogenase from Aspergillus niger (AnGDH) exhibits sigmoid 2-oxoglutarate saturation. Incubation with 2-hydroxyethyl disulfide (2-HED, the disulfide of 2-mercaptoethanol) resulted in preferential attenuation of AnGDH reductive amination (forward) activity but with a negligible effe...

Descripción completa

Detalles Bibliográficos
Autores principales: Walvekar, Adhish S., Choudhury, Rajarshi, Punekar, Narayan S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4079599/
https://www.ncbi.nlm.nih.gov/pubmed/24987966
http://dx.doi.org/10.1371/journal.pone.0101662
_version_ 1782323870960713728
author Walvekar, Adhish S.
Choudhury, Rajarshi
Punekar, Narayan S.
author_facet Walvekar, Adhish S.
Choudhury, Rajarshi
Punekar, Narayan S.
author_sort Walvekar, Adhish S.
collection PubMed
description NADP-Glutamate dehydrogenase from Aspergillus niger (AnGDH) exhibits sigmoid 2-oxoglutarate saturation. Incubation with 2-hydroxyethyl disulfide (2-HED, the disulfide of 2-mercaptoethanol) resulted in preferential attenuation of AnGDH reductive amination (forward) activity but with a negligible effect on oxidative deamination (reverse) activity, when monitored in the described standard assay. Such a disulfide modified AnGDH displaying less than 1.0% forward reaction rate could be isolated after 2-HED treatment. This unique forward inhibited GDH form (FIGDH), resembling a hypothetical ‘one-way’ active enzyme, was characterized. Kinetics of 2-HED mediated inhibition and protein thiol titrations suggested that a single thiol group is modified in FIGDH. Two site-directed cysteine mutants, C141S and C415S, were constructed to identify the relevant thiol in FIGDH. The forward activity of C141S alone was insensitive to 2-HED, implicating Cys141 in FIGDH formation. It was observed that FIGDH displayed maximal reaction rate only after a pre-incubation with 2-oxoglutarate and NADPH. In addition, compared to the native enzyme, FIGDH showed a four fold increase in K(0.5) for 2-oxoglutarate and a two fold increase in the Michaelis constants for ammonium and NADPH. With no change in the GDH reaction equilibrium constant, the FIGDH catalyzed rate of approach to equilibrium from reductive amination side was sluggish. Altered kinetic properties of FIGDH at least partly account for the observed apparent loss of forward activity when monitored under defined assay conditions. In sum, although Cys141 is catalytically not essential, its covalent modification provides a striking example of converting the biosynthetic AnGDH into a catabolic enzyme.
format Online
Article
Text
id pubmed-4079599
institution National Center for Biotechnology Information
language English
publishDate 2014
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-40795992014-07-08 Mixed Disulfide Formation at Cys141 Leads to Apparent Unidirectional Attenuation of Aspergillus niger NADP-Glutamate Dehydrogenase Activity Walvekar, Adhish S. Choudhury, Rajarshi Punekar, Narayan S. PLoS One Research Article NADP-Glutamate dehydrogenase from Aspergillus niger (AnGDH) exhibits sigmoid 2-oxoglutarate saturation. Incubation with 2-hydroxyethyl disulfide (2-HED, the disulfide of 2-mercaptoethanol) resulted in preferential attenuation of AnGDH reductive amination (forward) activity but with a negligible effect on oxidative deamination (reverse) activity, when monitored in the described standard assay. Such a disulfide modified AnGDH displaying less than 1.0% forward reaction rate could be isolated after 2-HED treatment. This unique forward inhibited GDH form (FIGDH), resembling a hypothetical ‘one-way’ active enzyme, was characterized. Kinetics of 2-HED mediated inhibition and protein thiol titrations suggested that a single thiol group is modified in FIGDH. Two site-directed cysteine mutants, C141S and C415S, were constructed to identify the relevant thiol in FIGDH. The forward activity of C141S alone was insensitive to 2-HED, implicating Cys141 in FIGDH formation. It was observed that FIGDH displayed maximal reaction rate only after a pre-incubation with 2-oxoglutarate and NADPH. In addition, compared to the native enzyme, FIGDH showed a four fold increase in K(0.5) for 2-oxoglutarate and a two fold increase in the Michaelis constants for ammonium and NADPH. With no change in the GDH reaction equilibrium constant, the FIGDH catalyzed rate of approach to equilibrium from reductive amination side was sluggish. Altered kinetic properties of FIGDH at least partly account for the observed apparent loss of forward activity when monitored under defined assay conditions. In sum, although Cys141 is catalytically not essential, its covalent modification provides a striking example of converting the biosynthetic AnGDH into a catabolic enzyme. Public Library of Science 2014-07-02 /pmc/articles/PMC4079599/ /pubmed/24987966 http://dx.doi.org/10.1371/journal.pone.0101662 Text en © 2014 Walvekar et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Walvekar, Adhish S.
Choudhury, Rajarshi
Punekar, Narayan S.
Mixed Disulfide Formation at Cys141 Leads to Apparent Unidirectional Attenuation of Aspergillus niger NADP-Glutamate Dehydrogenase Activity
title Mixed Disulfide Formation at Cys141 Leads to Apparent Unidirectional Attenuation of Aspergillus niger NADP-Glutamate Dehydrogenase Activity
title_full Mixed Disulfide Formation at Cys141 Leads to Apparent Unidirectional Attenuation of Aspergillus niger NADP-Glutamate Dehydrogenase Activity
title_fullStr Mixed Disulfide Formation at Cys141 Leads to Apparent Unidirectional Attenuation of Aspergillus niger NADP-Glutamate Dehydrogenase Activity
title_full_unstemmed Mixed Disulfide Formation at Cys141 Leads to Apparent Unidirectional Attenuation of Aspergillus niger NADP-Glutamate Dehydrogenase Activity
title_short Mixed Disulfide Formation at Cys141 Leads to Apparent Unidirectional Attenuation of Aspergillus niger NADP-Glutamate Dehydrogenase Activity
title_sort mixed disulfide formation at cys141 leads to apparent unidirectional attenuation of aspergillus niger nadp-glutamate dehydrogenase activity
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4079599/
https://www.ncbi.nlm.nih.gov/pubmed/24987966
http://dx.doi.org/10.1371/journal.pone.0101662
work_keys_str_mv AT walvekaradhishs mixeddisulfideformationatcys141leadstoapparentunidirectionalattenuationofaspergillusnigernadpglutamatedehydrogenaseactivity
AT choudhuryrajarshi mixeddisulfideformationatcys141leadstoapparentunidirectionalattenuationofaspergillusnigernadpglutamatedehydrogenaseactivity
AT punekarnarayans mixeddisulfideformationatcys141leadstoapparentunidirectionalattenuationofaspergillusnigernadpglutamatedehydrogenaseactivity