Role of phosphate-magnesium-binding regions in the high GTPase activity of rac1 protein.
rac1, a member of the low molecular mass GTP-binding protein family, has a 20-fold higher GTPase activity than H-ras, but the structural motifs responsible for this property do not appear to reside within the conserved amino acids of the consensus GTP-binding domains [Ménard, L., Tomhave, E., Casey, P.J., Uhing, R.J., Snyderman, R., & Disbury, J.R. (1992) Eur. J. Biochem 206, 537-546]. In this study, we determined the contribution, to the GTPase activity, of additional amino acids found in the phosphate-magnesium-binding (PM) regions of rac1. rac1 has three different amino acids from H-ras in each of the first two PM regions, while the third PM region is identical to that of H-ras. Mutation of the amino acids in the first PM region (aa 10-17) to the corresponding amino acids found in H-ras showed that modification of one of them, Asp11, resulted in a 50% decrease of the GTPase in rac1, whereas Gly13 and Ser17 had no effect. In the second PM region (aa 29-35), modification of the Pro29-Gly30 pair also reduced GTPase activity by 50% in rac1. rac1 mutated at positions 11 and 29 as well as 30 (P1-P2 mutant) had a 3-4-fold reduced GTPase activity compared to native rac1 (190 vs 552 pmol of GTP hydrolyzed/nmol of GTP gamma S-binding protein/min at 37 degrees C), suggesting a cooperative (but nonadditive) interaction between both domains. All mutants with reduced GTPase activity also had reduced affinity for GDP, shown by both equilibrium binding and GDP dissociation measurements.(ABSTRACT TRUNCATED AT 250 WORDS)
Ménard
L
L
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Snyderman
R
R
D000595:Amino Acid Sequence; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D020744:rac GTP-Binding Proteins
D010710:Phosphates; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins; D008274:Magnesium