Kinetics of interaction of nucleotides with nucleotide-free H-ras p21
Abstract
A method is described for the convenient preparation of substantial quantities of nucleotide-free p21 or of 1:1 complexes with nucleotides other than GDP. The nucleotide-free protein has been used for kinetic studies of the binding of GDP and GTP, making use of the fluorescent analogues 3'-(methylanthraniloyl)-2'-deoxy-GDP and -GTP. Stopped-flow studies have led to the formulation of a two-step binding mechanism for both GDP and GTP, involving initial rapid but weak binding of the nucleotide followed by a relatively slow (10-20 s-1 at 25 degrees C; 3-5 s-1 at 5 degrees C) quasi-irreversible isomerization reaction. By use of a nonequilibrium competition method, guanosine and GMP have been shown to interact weakly but significantly with p21 (dissociation constants of 153 and 29 microM, respectively). The presence of guanosine or GMP at the active site of p21 leads to a marked stabilization of p21 against spontaneous denaturation when compared with the nucleotide- and nucleoside-free protein.
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The difficulty of drugging KRAS reflects picomolar RAS affinity for GTP, high intracellular GTP concentration of about 500 nM, and the absence of a deep or pharmacologically actionable small-molecule-binding pocket; GTP-competitive RAS inhibitors are therefore unfeasible.
“The several-decade struggle to drug KRAS and other RAS isoforms25,61 reflects challenges posed by three key biochemical features: first, the picomolar affinity of RAS proteins for GTP62; second, the high intracellular concentrations of GTP (~500 nM)9; and third, the absence of a deep or pharmacologically actionable small-molecule-binding pocket in the RAS proteins.”
From The current state of the art and future trends in RAS-targeted cancer therapies (2022) · cites this paper as [62]
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“Early work on the GTP hydrolysis catalyzed by H-Ras and to a lesser extent by N-Ras was performed by using (32)P-labeled GTP at the -phosphate position, until the fluorescent N-methylanthraniloyl (mant) nucleotide derivatives became available in the early 1990s to measure the kinetics of hydrolysis and nucleotide exch…”
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Self-citationHenry, J. T., Coker, O., Chowdhury, S., Shen, J. P., Morris, V. K., Dasari, A., Raghav, K., Nusrat, M., Kee, B., Parseghian, C., et al. (2021) · JCO Precision Oncology · doi:10.1200/po.20.00256 · Cited by 66 · see citing works
- Authors
- John, J., Sohmen, R., Feuerstein, J., Linke, R., Wittinghofer, A., & Goody, R. S.
- Affiliations
- Max Planck Society (DE)
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 1990-06
- Volume
- 29
- Issue
- 25
- Pages
- 6058-6065
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- OpenAlex
- W2026128118
- Cited by
- 429
- References
- 0
- Citations by year
- 2026: 8 · 2025: 17 · 2024: 19 · 2023: 14 · 2022: 14 · 2021: 12 · 2020: 16 · 2019: 13 · 2018: 12 · 2017: 15 · 2016: 12 · 2015: 14 · 2014: 16 · 2013: 5 · 2012: 8
- FWCI
- 5.19
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Max Planck Digital Library (submittedVersion)
- MPG.PuRe (Max Planck Society) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Molecular Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
- Keywords
- p21ras, GDP binding, GTP binding, two-step binding mechanism, GTPase
- MeSH terms
- Guanosine, Guanosine Diphosphate, Guanosine Triphosphate, Kinetics, Nucleotides, Spectrometry, Fluorescence, Oncogene Protein p21(ras), ortho-Aminobenzoates
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