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  The role of the metal ion in the p21rascatalysed GTP-hydrolysis: Mn2+ versus Mg2+

Schweins, T., Scheffzek, K., Assheuer, R., & Wittinghofer, A. (1997). The role of the metal ion in the p21rascatalysed GTP-hydrolysis: Mn2+ versus Mg2+. Journal of Molecular Biology (London), 266(4), 847-856. doi:10.1006/jmbi.1996.0814.

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Genre: Journal Article
Alternative Title : The role of the metal ion in the p21ras catalysed GTP-hydrolysis: Mn2+versus Mg2+ 1

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JMolBiol_266_1997_847.pdf (Any fulltext), 560KB
 
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 Creators:
Schweins, Thomas, Author
Scheffzek, Klaus1, Author           
Assheuer, Ralf, Author
Wittinghofer, Alfred1, Author           
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1Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society, ou_1497712              

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Free keywords: Ras-p21; GTP hydrolysis; 3D structure; metal ion; manganese
 Abstract: GTP and ATP hydrolysing proteins have an absolute requirement for a divalent cation, which is usually Mg2+, as a cofactor in the enzymatic reaction. Other phosphoryl transfer enzymes employ more than one divalent ion for the enzymatic reaction. It is shown here for p21ras, a well studied example of GTP hydrolysing proteins, that GTP and ATP hydrolysing proteins have an absolute requirement for a divalent cation, which is usually Mg2+, as a cofactor in the enzymatic reaction. Other phosphoryl transfer enzymes employ more than one divalent ion for the enzymatic reaction. It is shown here for p21ras, a well studied example of GTP hydrolysing proteins, that the GTP-hydrolysis rate is significantly faster if Mg2+ is replaced by Mn2+, both in the presence or absence of its GTPase-activating protein Ras-GAP. This effect is not due to a different stoichiometry of metal ion binding, since one metal ion is sufficient for full enzymatic activity. To determine the role of the metal ion, the crystal structure of p21(G12P)·GppCp complexed with Mn2+ was determined and shown to be very similar to the corresponding p21(G12P)·GppCp·Mg2+ structure. Especially the coordination sphere around the metal ions is very similar, and no second metal ion binding site could be detected, consistent with the assumption that one metal ion is sufficient for GTP hydrolysis. In order to explain the biochemical differences, we analysed the GTPase reaction mechanism with a linear free energy relationships approach. The result suggests that the reaction mechanism is not changed with Mn2+ but that the transition metal ion Mn2+ shifts the pKa of the γ-phosphate by almost half a unit and increases the reaction rate due to an increase in the basicity of GTP acting as the general base. This suggests that the intrinsic GTPase reaction could be an attractive target for anti-cancer drug design. By using Rap1A and Ran, we show that the acceleration of the GTPase by Mn2+ appears to be a general phenomenon of GTP-binding proteins

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Language(s): eng - English
 Dates: 1996-07-311996-11-281997-03-07
 Publication Status: Published in print
 Pages: 10
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Degree: -

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Title: Journal of Molecular Biology (London)
  Other : J Mol Biol
Source Genre: Journal
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Publ. Info: London : Academic Press
Pages: - Volume / Issue: 266 (4) Sequence Number: - Start / End Page: 847 - 856 Identifier: ISSN: 0022-2836
CoNE: https://pure.mpg.de/cone/journals/resource/954922646042