MECHANISM OF GTP HYDROLYSIS BY P21(N-RAS) CATALYZED BY GAP - STUDIES WITH A FLUORESCENT GTP ANALOG

The mechanism of the hydrolysis of GTP by p21N-ras and its activation by the catalytic domain of p120 GTPase activating protein (GAP) have b een studied using a combination of chemical and fluorescence measureme nts with the fluorescent GTP analogue, 2'(3')-O-(N-methylanthraniloyl) GTP (mantGTP). Since the concentration of active p21 is important in t hese measurements, various assays for both total protein and active p2 1 were investigated. All assays gave good agreement except the filter binding assay of [H-3]-GDP bound to p21, which gave values of 35-40% c ompared to the other methods. Concentrations of p21 were thus based on the absorbance of the mant-chromophore of the p21.mant-nucleotide com plexes. The rate constants of the elementary steps of the p21 intrinsi c GTPase activity and the GAP activated activity were similar between GTP and mantGTP. Incubation of a stoichiometric complex of p21.mantGTP results in a biphasic decrease in fluorescence. The second phase occu rs with the same rate constant as the cleavage step and is accelerated by GAP. No other steps of the mechanism are affected by GAP. Incubati on of a stoichiometric complex of p21.mantGpp[NH]p also results in a b iphasic decrease in fluorescence even though cleavage does not occur. This is interpreted that the cleavage step of p21.GTP is preceded by a nd controlled by an isomerization of the p21.GTP complex. GAP accelera tes the rate constant of the second fluorescence phase occurring with p21.mantGpp[NH]p. This result shows that GAP accelerates the proposed isomerization which limits GTP cleavage rather than the cleavage step itself.