Biochemical properties of Ha-ras encoded p21 mutants and mechanism of the autophosphorylation reaction.
Kinetic studies performed on p21H guanine nucleotide complexes with and without Mg2+ show that point mutations at positions 12, 59, and 61 each have a different effect on the rate of nucleotide dissociation. Double mutants with a combination of these amino acid substitutions reveal that the effects of each mutation on these kinetics are interactive (nonadditive) for positions 12 and 59 and approximately additive for the positions 12 and 61. The magnitude and direction of the effects seen are dependent on the nature of the nucleotide and whether or not the complexes contain Mg2+. All the mutants have reduced GTPase activity. It is also shown that the autophosphorylation reaction velocity is of first order with respect to the protein concentration and that this reaction is an intramolecular one, which takes place as a side reaction of the GTPase reaction. The autophosphorylation is not reversible under the experimental conditions. The covalently bound phosphate does not decrease the nucleotide-binding ability of the protein nor does it change the relative affinity of the protein for GTP versus GDP. The results are discussed in terms of the structural model and function of p21H.
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009154:Mutation; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras); D013329:Structure-Activity Relationship
D006150:Guanine Nucleotides; D010710:Phosphates; D011518:Proto-Oncogene Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D016283:Proto-Oncogene Proteins p21(ras); D008274:Magnesium