Transient kinetic studies on the interaction of Ras and the Ras-binding domain of c-Raf-1 reveal rapid equilibration of the complex.
Transient kinetic methods have been used to analyze the interaction between the Ras-binding domain (RBD) of c-Raf-1 and a complex of H-Ras and a GTP analogue. The results obtained show that the binding is a two-step process, with an initial rapid equilibrium step being followed by an isomerization reaction occurring at several hundred per second. The reversal of this step determines the rate constant for dissociation, which is on the order of 10 s-1. The lifetime of the complex is therefore on the order of 50-100 ms, which is much shorter than the lifetime of GTP at the active site of H-Ras as determined by the intrinsic GTPase reaction. This suggests that multiple interactions of a single activated Ras molecule and Raf can occur, the number being limited by the competing interaction with GAP. The GDP complex of H-Ras binds more than 2 orders of magnitude more weakly than the GTP-analogue complex, mainly due to a significant weakening of the initial binding equilibrium reaction in the GDP state, thereby avoiding even short-lived recruitment of Raf to the plasma membrane by the inactive Ras form.
Abteilung Physikalische Biochemie, Max-Planck-Institut für Molekulare Physiologie, Dortmund, Germany.
J R
JR
Sydor
M
M
Engelhard
A
A
Wittinghofer
R S
RS
Goody
C
C
Herrmann
D005456:Fluorescent Dyes; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000235:genetics; D014443:Tyrosine / Q000235:genetics; D062367:ortho-Aminobenzoates / Q000378:metabolism; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
C096262:3'-O-(N-methylanthraniloyl)-2'-deoxyguanosine 5'-(imido)triphosphate; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D062367:ortho-Aminobenzoates; D006153:Guanosine Diphosphate; D014443:Tyrosine; D006160:Guanosine Triphosphate; D014364:Tryptophan; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins