The conserved arginine in rho-GTPase-activating protein is essential for efficient catalysis but not for complex formation with Rho.GDP and aluminum fluoride.
The Rho family of small GTP-binding proteins are downregulated by an intrinsic GTPase, which is enhanced by GTPase-activating proteins (GAPs). RhoGAPs contain a single conserved arginine residue that has been proposed to be involved in catalysis. Here, the role of this arginine has been elucidated by mutagenesis followed by determination of catalytic and equilibrium binding constants using single-turnover kinetics, isothermal titration calorimetry, and scintillation proximity assays. The turnover numbers for wild-type, R282A, and R282K RhoGAPs were 5.4, 0.023, and 0.010 s-1, respectively. Thus, the function of this arginine could not be replaced by lysine or alanine. Nevertheless, the R282A mutation had a minimal effect on the binding affinity of RhoGAP for either Rho. GTP or Rho.GMPPNP, which confirms the importance of the arginine residue for catalysis as opposed to formation of the protein-protein complex. The R282A mutant RhoGAP still increased the hydrolysis rate of Rho.GTP by 160-fold, whereas the wild-type enzyme increased it by 38000-fold. We conclude that this arginine contributes half of the total reduction of activation energy of catalysis. In the presence of aluminum fluoride, the R282A mutant RhoGAP binds almost as well as the wild type to Rho.GDP, demonstrating that the conserved arginine is not required for this interaction. The affinity of wild-type RhoGAP for the triphosphate form of Rho is similar to that for Rho.GDP with aluminum fluoride. These last two observations show that this complex is not associated with the free energy changes expected for the transition state, although the Rho.GDP.AlF4-.RhoGAP complex might well be a close structural approximation.
Graham
D L
DL
Division of Physical Biochemistry, National Institute of Medical Research, London, U.K.
Eccleston
J F
JF
Lowe
P N
PN
D000409:Alanine / Q000235:genetics; D017607:Aluminum Compounds / Q000378:metabolism*; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D017124:Conserved Sequence; D005459:Fluorides / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000235:genetics; D046911:Macromolecular Substances; D011485:Protein Binding / Q000235:genetics; D012234:Rho Factor / Q000235:genetics / Q000378:metabolism*; D062367:ortho-Aminobenzoates / Q000378:metabolism
D017607:Aluminum Compounds; D020690:GTPase-Activating Proteins; D046911:Macromolecular Substances; D012234:Rho Factor; D062367:ortho-Aminobenzoates; C085379:rho GTPase-activating protein; C065197:3'-(methylanthraniloyl)-2'-deoxy-guanosine diphosphate; D006153:Guanosine Diphosphate; D006165:Guanylyl Imidodiphosphate; D001120:Arginine; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D008239:Lysine; D000409:Alanine; D005459:Fluorides; C032311:aluminum fluoride