The role of the metal ion in the p2l(ras) catalysed GTP-hydrolysis: Mn2+ versus Mg2+

被引:55
作者
Schweins, T [1 ]
Scheffzek, K [1 ]
Assheuer, R [1 ]
Wittinghofer, A [1 ]
机构
[1] MAX PLANCK INST MOL PHYSIOL, D-44139 DORTMUND, GERMANY
关键词
Ras-p21; GTP hydrolysis; 3D structure; metal ion; manganese;
D O I
10.1006/jmbi.1996.0814
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 p21(ras) a well skudied 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 pK(a) of the gamma-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. (C) 1997 Academic Press Limited.
引用
收藏
页码:847 / 856
页数:10
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