Insight into temperature dependence of GTPase activity in human guanylate binding protein-1.
Interferon-γ induced human guanylate binding protein-1(hGBP1) belongs to a family of dynamin related large GTPases. Unlike all other GTPases, hGBP1 hydrolyzes GTP to a mixture of GDP and GMP with GMP being the major product at 37°C but GDP became significant when the hydrolysis reaction was carried...
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doaj-1bf00f209b79492e9a9506fcb73dfc1e2020-11-25T00:12:33ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-0177e4048710.1371/journal.pone.0040487Insight into temperature dependence of GTPase activity in human guanylate binding protein-1.Anjana RaniEsha PanditaSafikur RahmanShashank DeepApurba Kumar SauInterferon-γ induced human guanylate binding protein-1(hGBP1) belongs to a family of dynamin related large GTPases. Unlike all other GTPases, hGBP1 hydrolyzes GTP to a mixture of GDP and GMP with GMP being the major product at 37°C but GDP became significant when the hydrolysis reaction was carried out at 15°C. The hydrolysis reaction in hGBP1 is believed to involve with a number of catalytic steps. To investigate the effect of temperature in the product formation and on the different catalytic complexes of hGBP1, we carried out temperature dependent GTPase assays, mutational analysis, chemical and thermal denaturation studies. The Arrhenius plot for both GDP and GMP interestingly showed nonlinear behaviour, suggesting that the product formation from the GTP-bound enzyme complex is associated with at least more than one step. The negative activation energy for GDP formation and GTPase assay with external GDP together indicate that GDP formation occurs through the reversible dissociation of GDP-bound enzyme dimer to monomer, which further reversibly dissociates to give the product. Denaturation studies of different catalytic complexes show that unlike other complexes the free energy of GDP-bound hGBP1 decreases significantly at lower temperature. GDP formation is found to be dependent on the free energy of the GDP-bound enzyme complex. The decrease in the free energy of this complex at low temperature compared to at high is the reason for higher GDP formation at low temperature. Thermal denaturation studies also suggest that the difference in the free energy of the GTP-bound enzyme dimer compared to its monomer plays a crucial role in the product formation; higher stability favours GMP but lower favours GDP. Thus, this study provides the first thermodynamic insight into the effect of temperature in the product formation of hGBP1.http://europepmc.org/articles/PMC3394710?pdf=render |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Anjana Rani Esha Pandita Safikur Rahman Shashank Deep Apurba Kumar Sau |
spellingShingle |
Anjana Rani Esha Pandita Safikur Rahman Shashank Deep Apurba Kumar Sau Insight into temperature dependence of GTPase activity in human guanylate binding protein-1. PLoS ONE |
author_facet |
Anjana Rani Esha Pandita Safikur Rahman Shashank Deep Apurba Kumar Sau |
author_sort |
Anjana Rani |
title |
Insight into temperature dependence of GTPase activity in human guanylate binding protein-1. |
title_short |
Insight into temperature dependence of GTPase activity in human guanylate binding protein-1. |
title_full |
Insight into temperature dependence of GTPase activity in human guanylate binding protein-1. |
title_fullStr |
Insight into temperature dependence of GTPase activity in human guanylate binding protein-1. |
title_full_unstemmed |
Insight into temperature dependence of GTPase activity in human guanylate binding protein-1. |
title_sort |
insight into temperature dependence of gtpase activity in human guanylate binding protein-1. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2012-01-01 |
description |
Interferon-γ induced human guanylate binding protein-1(hGBP1) belongs to a family of dynamin related large GTPases. Unlike all other GTPases, hGBP1 hydrolyzes GTP to a mixture of GDP and GMP with GMP being the major product at 37°C but GDP became significant when the hydrolysis reaction was carried out at 15°C. The hydrolysis reaction in hGBP1 is believed to involve with a number of catalytic steps. To investigate the effect of temperature in the product formation and on the different catalytic complexes of hGBP1, we carried out temperature dependent GTPase assays, mutational analysis, chemical and thermal denaturation studies. The Arrhenius plot for both GDP and GMP interestingly showed nonlinear behaviour, suggesting that the product formation from the GTP-bound enzyme complex is associated with at least more than one step. The negative activation energy for GDP formation and GTPase assay with external GDP together indicate that GDP formation occurs through the reversible dissociation of GDP-bound enzyme dimer to monomer, which further reversibly dissociates to give the product. Denaturation studies of different catalytic complexes show that unlike other complexes the free energy of GDP-bound hGBP1 decreases significantly at lower temperature. GDP formation is found to be dependent on the free energy of the GDP-bound enzyme complex. The decrease in the free energy of this complex at low temperature compared to at high is the reason for higher GDP formation at low temperature. Thermal denaturation studies also suggest that the difference in the free energy of the GTP-bound enzyme dimer compared to its monomer plays a crucial role in the product formation; higher stability favours GMP but lower favours GDP. Thus, this study provides the first thermodynamic insight into the effect of temperature in the product formation of hGBP1. |
url |
http://europepmc.org/articles/PMC3394710?pdf=render |
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