Ras-mediated cleavage of a GTP analogue by a novel mechanism

The small guanosine triphosphate (GTP) binding protein Ras is involved in m any cellular signal transduction processes leading to cell growth, differen tiation and apoptosis. Mutations in ras genes are found in a large number o f human tumours. GTP hydrolysis, the process that normally leads to the tra nsition 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(DASP)-phosphoramidate-GTP, a sub strate for GTP-binding proteins, enables switching to the inactive GDP form in both wild-type and oncogenic Ras. Here we show by HPLC, mass spectromet ry and NMR spectroscopy that the mechanism of this DABP-GTPase reaction is different from the physiological GTPase reaction. The gamma -phosphate grou p is not attacked by a nucleophilic water molecule, but rather by,the aroma tic amino group of the analogue, which leads to the generation of a stable cyclic diamidate product, These findings, hove potential implications for t he development of anti-Ras drugs.