Ras-mediated cleavage of a GTP analogue by a novel mechanism.
The small guanosine triphosphate (GTP) binding protein Ras is involved in many cellular signal transduction processes leading to cell growth, differentiation and apoptosis. Mutations in ras genes are found in a large number of human tumours. GTP hydrolysis, the process that normally leads to the transition of the Ras protein from the active (GTP-bound) form to the inactive (GDP-bound) form is impaired due to these oncogenic mutations. In contrast, the GTP analogue 3,4-diaminobenzophenone(DABP)-phosphoramidate-GTP, a substrate for GTP-binding proteins, enables switching to the inactive GDP form in both wild-type and oncogenic Ras. Here we show by HPLC, mass spectrometry and NMR spectroscopy that the mechanism of this DABP-GTPase reaction is different from the physiological GTPase reaction. The gamma-phosphate group is not attacked by a nucleophilic water molecule, but rather by the aromatic amino group of the analogue, which leads to the generation of a stable cyclic diamidate product. These findings have potential implications for the development of anti-Ras drugs.
Gail
R
R
Max-Planck-Institut für molekulare Physiologie, Abteilung Strukturelle Biologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Costisella
B
B
Ahmadian
M R
MR
Wittinghofer
A
A
D001577:Benzophenones / Q000737:chemistry* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D018631:ras Proteins / Q000737:chemistry* / Q000378:metabolism
C109051:3,4-diaminobenzophenone-phosphonoamidate-GTP; D001577:Benzophenones; D006160:Guanosine Triphosphate; D018631:ras Proteins