Mechanism of GTP hydrolysis by p21N-ras catalyzed by GAP: studies with a fluorescent GTP analogue.
The mechanism of the hydrolysis of GTP by p21N-ras and its activation by the catalytic domain of p120 GTPase activating protein (GAP) have been studied using a combination of chemical and fluorescence measurements with the fluorescent GTP analogue, 2'(3')-O-(N-methylanthraniloyl)GTP (mantGTP). Since the concentration of active p21 is important in these measurements, various assays for both total protein and active p21 were investigated. All assays gave good agreement except the filter binding assay of [3H]-GDP bound to p21, which gave values of 35-40% compared to the other methods. Concentrations of p21 were thus based on the absorbance of the mant-chromophore of the p21-mant-nucleotide complexes. The rate constants of the elementary steps of the p21 intrinsic 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 occurs with the same rate constant as the cleavage step and is accelerated by GAP. No other steps of the mechanism are affected by GAP. Incubation of a stoichiometric complex of p21.mantGpp[NH]p also results in a biphasic decrease in fluorescence even though cleavage does not occur. This is interpreted that the cleavage step of p21.GTP is preceded by and controlled by an isomerization of the p21.GTP complex. GAP accelerates 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.
Moore
K J
KJ
Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London, U.K.
Webb
M R
MR
Eccleston
J F
JF
D002850:Chromatography, Gel; D004926:Escherichia coli; D005456:Fluorescent Dyes; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020690:GTPase-Activating Proteins; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D009994:Osmolar Concentration; D011506:Proteins / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D062367:ortho-Aminobenzoates / Q000378:metabolism; D020703:ras GTPase-Activating Proteins
D005456:Fluorescent Dyes; D020690:GTPase-Activating Proteins; D011506:Proteins; D062367:ortho-Aminobenzoates; D020703:ras GTPase-Activating Proteins; D006153:Guanosine Diphosphate; D006165:Guanylyl Imidodiphosphate; C063981:2'(3')-O-(N-methyl)anthraniloylguanosine 5'-triphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D016283:Proto-Oncogene Proteins p21(ras)