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Study of Ras catalytic mechanism of intrinsic hydrolysis of GTP
Author(s) -
Knihtila Ryan,
Holzapfel Genevieve,
Mattos Carla
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.831.10
Subject(s) - allosteric regulation , gtp' , chemistry , active site , molecule , gtpase , effector , stereochemistry , catalysis , crystallography , biophysics , enzyme , biochemistry , biology , organic chemistry
Ras cycles between an active GTP bound form and an inactive GDP bound form. While Ras hydrolysis of GTP to GDP is intrinsically slow, we have discovered a GAP‐independent allosteric switch mechanism that results in ordering of switch II, placing the catalytic Q61 in the active site. This is expected to enhance the rate of hydrolysis through a mechanism where GTP acts as the general base, abstracting a proton from a nucleophilic water molecule, and shuttling it to a bridging water molecule that donates H‐bonds to Y32 in switch I and Q61 in switch II. This would promote a partial positive charge on the bridging water molecule, analogous to the arginine finger of GAP. In order to validate this mechanism we are solving the neutron crystal structure of H‐Ras to reveal the positions of hydrogen atoms in the active site. We have shown that perdeuteration of Ras results in an unchanged active site and have optimized crystal growth for neutron crystallography. We are also doing a series of hydrolysis experiments on wild type and mutant H‐Ras in the presence of NORE1A, an effector that stabilizes the active site in the same conformation seen with an activated allosteric switch in the presence of Raf‐RBD. A rate decrease in RasY32F will establish this residue as important in catalysis, while the RasQ61L mutant is expected to decrease the rate of reaction in the presence of NOR1EA as it does in the absence of effector proteins.