LINEAR FREE-ENERGY RELATIONSHIPS IN THE INTRINSIC AND GTPASE-ACTIVATING PROTEIN-STIMULATED GUANOSINE 5'-TRIPHOSPHATE HYDROLYSIS OF P21(RAS)

Controlling the hydrolysis rate of GTP bound to guanine nucleotide bin ding proteins is crucial for the right timing of many biological proce sses. Theoretical, structural, and functional studies have demonstrate d that in p21(ras) the substrate of the reaction, GTP itself, plays a central role by acting as the base catalyst. This substrate-assisted r eaction mechanism was analyzed with the help of linear free energy rel ationships (LFERs). Here we present experimental data that further sup port the proposed mechanism. We extend the LFER analysis to a wide ran ge of oncogenic as well as nontransforming Pas mutants, It is illustra ted that almost all Pas variants follow the observed LFER and thus als o the same reaction path. Further, the reduced GTPase reaction rate th at characterizes the oncogenic effect of many of the p21 mutants found in human tumors seems to be a consequence of a slightly reduced pK(a) of the gamma-phosphate group of bound GTP. Factors causing a pK(a) de viation of just 0.5 unit are enough to slow the intrinsic GTPase react ion rate significantly, and the system may exhibit as a consequence of this an oncogenic potential. Interestingly, we also found oncogenic m utations that do not follow the regular LFER. This suggests that the o ncogenic effect of distinct Ras mutants has a different physical origi n. The results presented might aid in the design of drugs aimed at rea ctivating the GTPase reaction of many oncogenic p21(ras) mutants. We a lso analyzed the stimulated GTPase reaction of p21(ras) by the GTPase activating protein (GAP) and the GTPase reaction of Rap1A, a Pas-relat ed GTP binding protein, with similar approaches, The corresponding res ults indicate that the GAP-stimulated GTPase as well as the Rap1A-cata lyzed reaction seem to follow the same substrate-assisted reaction mec hanism. However, the correlation coefficient for the GAP-catalyzed rea ction is different from the corresponding coefficient for the intrinsi c reaction. While the intrinsic reaction exhibits a Bronsted slope of beta = 2.1, the corresponding value for the GAP-activated reaction is beta = 4.9.