GTP hydrolysis mechanisms in ras p21 and in the ras-GAP complex studied by fluorescence measurements on tryptophan mutants.
We have substituted leucine 56 or tyrosine 64 of p21 ras with a tryptophan. The intrinsic fluorescence of this tryptophan was used as an internal conformational probe for time-resolved biochemical studies of the ras protein. The slow intrinsic GTPase, GDP/GTP exchange induced by the SDC25 "exchange factor", and the fast GTP hydrolysis induced by GAP were studied. Tryptophan fluorescence of mutated ras is very sensitive to magnesium binding, GDP/GTP exchange, and GTP hydrolysis (changes in tyrosine fluorescence of wild-type ras are also observed but with a lower sensitivity). Nucleotide affinities, exchange kinetics, and intrinsic GTPase rates of the mutated ras could be measured by this method and were found to be close to those of wild-type ras. The SDC25 gene product enhances GDP/GTP exchange in both mutants. In both mutants, a slow fluorescence change follows the binding of GTP gamma S; its kinetics are close to those of the intrinsic GTPase, suggesting that a slow conformational change precedes the GTPase and is the rate-limiting step, as proposed by Neal et al. (1990) (Proc. Natl. Acad. Sci. U.S.A. 87, 3562-3565). GAP interacts with both mutant ras proteins and accelerates the GTPase of (L56W)ras but not that of (Y64W)ras, suggesting a role for tyrosine 64 in GAP-induced GTP hydrolysis. However, GAP does not accelerate the slow conformational change following GTP gamma S binding in either of the mutated ras proteins. This suggests that the fast GAP-induced catalysis of GTP hydrolysis that is observed with (L56W)ras bypasses the slow conformational change associated with the intrinsic GTPase and therefore might proceed by a different mechanism.
Antonny
B
B
CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France.
Chardin
P
P
Roux
M
M
Chabre
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005656:Fungal Proteins / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins; D020690:GTPase-Activating Proteins; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015689:Oncogene Protein p21(ras) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D011506:Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics*; D020668:rap GTP-Binding Proteins; D020703:ras GTPase-Activating Proteins
D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D014364:Tryptophan; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D015689:Oncogene Protein p21(ras); D020668:rap GTP-Binding Proteins; D008274:Magnesium