Monitoring the GAP catalyzed H-Ras GTPase reaction at atomic resolution in real time.

Allin C; Ahmadian MR; Wittinghofer AGerwert K

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Monitoring the GAP catalyzed H-Ras GTPase reaction at atomic resolution in real time.

机译：Monitoring the GAP catalyzed H-Ras GTPase reaction at atomic resolution in real time.

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摘要

The molecular reaction mechanism of the GTPase-activating protein (GAP)-catalyzed GTP hydrolysis by Ras was investigated by time resolved Fourier transform infrared (FTIR) difference spectroscopy using caged GTP (P(3)-1-(2-nitro)phenylethyl guanosine 5'-O-triphosphate) as photolabile trigger. This approach provides the complete GTPase reaction pathway with time resolution of milliseconds at the atomic level. Up to now, one structural model of the GAP x Ras x GDP x AlF(x) transition state analog is known, which represents a "snap shot" along the reaction-pathway. As now revealed, binding of GAP to Ras x GTP shifts negative charge from the gamma to beta phosphate. Such a shift was already identified by FTIR in GTP because of Ras binding and is now shown to be enhanced by GAP binding. Because the charge distribution of the GAP x Ras x GTP complex thus resembles a more dissociative-like transition state and is more like that in GDP, the activation free energy is reduced. An intermediate is observed on the reaction pathway that appears when the bond between beta and gamma phosphate is cleaved. In the intermediate, the released P(i) is strongly bound to the protein and surprisingly shows bands typical of those seen for phosphorylated enzyme intermediates. All these results provide a mechanistic picture that is different from the intrinsic GTPase reaction of Ras. FTIR analysis reveals the release of P(i) from the protein complex as the rate-limiting step for the GAP-catalyzed reaction. The approach presented allows the study not only of single proteins but of protein-protein interactions without intrinsic chromophores, in the non-crystalline state, in real time at the atomic level.

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《Proceedings of the National Academy of Sciences of the United States of America》 |2001年第14期|7754-7759|共6页
作者
Allin C; Ahmadian MR; Wittinghofer AGerwert K;
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作者单位

Lehrstuhl fur Biophysik, Ruhr-Universitat Bochum, D-44780 Bochum, Germany.;

hylo.life.uiuc.edu;

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收录信息美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
原文格式 PDF
正文语种英语
中图分类自然科学总论;
关键词
GTPase Activating Proteins metabolism; Proto Oncogene Protein p21ras metabolism;

Monitoring the GAP catalyzed H-Ras GTPase reaction at atomic resolution in real time.

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