Structural analysis of the Ras GTPase activating protein catalytic domain by semirandom mutagenesis: implications for a mechanism of interaction with Ras-GTP.
The bovine complementary DNA encoding the catalytic domain of Ras GTPase activating protein was mutagenized semirandomly using a variation of the polymerase chain reaction. Sixty-four mutated codons were identified with seventeen of the mutations deleterious to Ras GTPase activating function. All of the inactivating single mutations affected the structure of the catalytic fragment as assessed by large decreases in soluble protein when expressed in Escherichia coli. Upon examination of the Ras binding properties of 10 of the mutants, only 1 was measurably impaired for Ras binding and 4 appeared to have increased affinity for Ras. These results demonstrate that Ras binding and GTPase activation are two distinct properties of GTPase activating protein. Additionally, the catalytic mechanism of GTPase activating protein is much more sensitive to structural perturbation than is Ras binding.
Hettich
L
L
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
Marshall
M
M
D000595:Amino Acid Sequence; D003062:Codon / Q000235:genetics*; D020690:GTPase-Activating Proteins; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics*; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D020703:ras GTPase-Activating Proteins
D003062:Codon; D020690:GTPase-Activating Proteins; D011506:Proteins; D020703:ras GTPase-Activating Proteins