Mutational and kinetic analyses of the GTPase-activating protein (GAP)-p21 interaction: the C-terminal domain of GAP is not sufficient for full activity.
The GTPase-activating protein (GAP) stimulates the GTPase reaction of p21 by 5 orders of magnitude such that the kcat of the reaction is increased to 19 s-1. Mutations of residues in loop L1 (Gly-12 and Gly-13), in loop L2 (Thr-35 and Asp-38), and in loop L4 (Gln-61 and Glu-63) influence the reaction in different ways, but all of these mutant p21 proteins still form complexes with GAP. The C-terminal domain of the human GAP gene product, GAP334, which comprises residues 714 to 1047, is 20 times less active than full-length GAP on a molar basis and has a fourfold lower affinity. This finding indicates that the N terminus of GAP containing the SH2 domains modifies the interaction between the catalytic domain and p21.
Gideon
P
P
Max-Planck-Institut für medizinische Forschung, Abteilung Biophysik, Heidelberg, Germany.
John
J
J
Frech
M
M
Lautwein
A
A
Clark
R
R
Scheffler
J E
JE
Wittinghofer
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002460:Cell Line; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D020690:GTPase-Activating Proteins; D005822:Genetic Vectors; D006160:Guanosine Triphosphate / Q000378:metabolism; D007313:Insecta; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016716:PC12 Cells; D011487:Protein Conformation; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D020703:ras GTPase-Activating Proteins
D000911:Antibodies, Monoclonal; D020690:GTPase-Activating Proteins; D011506:Proteins; D011994:Recombinant Proteins; D020703:ras GTPase-Activating Proteins; D006160:Guanosine Triphosphate; D016283:Proto-Oncogene Proteins p21(ras)