The mechanism of Ras GTPase activation by neurofibromin.
Individual rate constants have been determined for each step of the Ras.GTP hydrolysis mechanism, activated by neurofibromin. Fluorescence intensity and anisotropy stopped-flow measurements used the fluorescent GTP analogue, mantGTP (2'(3')-O-(N-methylanthraniloyl)GTP), to determine rate constants for binding and release of neurofibromin. Quenched flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor, MDCC-PBP was used to measure phosphate release kinetics. Phosphate-water oxygen exchange, using (18)O-substituted GTP and inorganic phosphate (P(i)), was used to determine the extent of reversal of the hydrolysis step and of P(i) binding. The data show that neurofibromin and P(i) dissociate from the NF1.Ras.GDP.P(i) complex with identical kinetics, which are 3-fold slower than the preceding cleavage step. A model is presented in which the P(i) release is associated with the change of Ras from "GTP" to "GDP" conformation. In this model, the conformation change on P(i) release causes the large change in affinity of neurofibromin, which then dissociates rapidly.
Phillips
Robert A
RA
National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.
Hunter
Jackie L
JL
Eccleston
John F
JF
Webb
Martin R
MR
D001665:Binding Sites; D002384:Catalysis; D003374:Coumarins / Q000378:metabolism; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D025542:Neurofibromin 1 / Q000378:metabolism / Q000494:pharmacology*; D010100:Oxygen / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D062367:ortho-Aminobenzoates / Q000378:metabolism*
D003374:Coumarins; D025542:Neurofibromin 1; D010710:Phosphates; D062367:ortho-Aminobenzoates; D006153:Guanosine Diphosphate; C088410:N-(2-(1-maleimidyl)ethyl)-7-(diethylamino)coumarin-3-carboxamide; C063981:2'(3')-O-(N-methyl)anthraniloylguanosine 5'-triphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D010100:Oxygen