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Title
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Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis.
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Authors
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E.F.Pai,
U.Krengel,
G.A.Petsko,
R.S.Goody,
W.Kabsch,
A.Wittinghofer.
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Ref.
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Embo J, 1990,
9,
2351-2359.
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PubMed id
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Abstract
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The crystal structure of the H-ras oncogene protein p21 complexed to the slowly
hydrolysing GTP analogue GppNp has been determined at 1.35 A resolution. 211
water molecules have been built into the electron density. The structure has
been refined to a final R-factor of 19.8% for all data between 6 A and 1.35 A.
The binding sites of the nucleotide and the magnesium ion are revealed in high
detail. For the stretch of amino acid residues 61-65, the temperature factors of
backbone atoms are four times the average value of 16.1 A2 due to the multiple
conformations. In one of these conformations, the side chain of Gln61 makes
contact with a water molecule, which is perfectly placed to be the nucleophile
attacking the gamma-phosphate of GTP. Based on this observation, we propose a
mechanism for GTP hydrolysis involving mainly Gln61 and Glu63 as activating
species for in-line attack of water. Nucleophilic displacement is facilitated by
hydrogen bonds from residues Thr35, Gly60 and Lys16. A mechanism for rate
enhancement by GAP is also proposed.
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