Switching nucleotide specificity of Ha-Ras p21 by a single amino acid substitution at aspartate 119.
We examined c-Ha-Ras harboring an aspartate to asparagine substitution at position 119 (mutation D119N). The Asp-119 is part of the conserved NKXD motif shared by members of the regulatory GTPase family. This asparagine residue has been proposed to participate in direct bonding to the guanine ring and to determine the guanine-nucleotide binding specificity. The D119N mutation was found to alter nucleotide specificity of Ha-Ras from guanine to xanthine, an observation that directly supports the essential role of hydrogen bonding between the side chain of the aspartic acid residue and the guanine ring in nucleotide binding specificity. Besides nucleotide binding specificity, the D119N mutation has little or no effect on the interaction of Ha-Ras with SDC25C, SOS1, GAP, or Raf. Neither does it affect the hydrolysis of nucleotide triphosphate. Like xanthine-nucleotide-specific EF-Tu, xanthine-nucleotide-specific Ras and related proteins will be useful tools for elucidating cellular systems containing multiple regulatory GTPases.
Zhong
J M
JM
Molecular Biology Department, New York State Institute for Basic Research in Developmental Disabilities, Staten Island 10314, USA.
Chen-Hwang
M C
MC
Hwang
Y W
YW
D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D019204:GTP-Binding Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics; D020668:rap GTP-Binding Proteins; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D009711:Nucleotides; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D011518:Proto-Oncogene Proteins; D020703:ras GTPase-Activating Proteins; D001224:Aspartic Acid; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D019204:GTP-Binding Proteins; D016283:Proto-Oncogene Proteins p21(ras); D020668:rap GTP-Binding Proteins