Transition state structures and the roles of catalytic residues in GAP-facilitated GTPase of Ras as elucidated by (18)O kinetic isotope effects.
Ras-catalyzed guanosine 5' triphosphate (GTP) hydrolysis proceeds through a loose transition state as suggested in our previous study of (18)O kinetic isotope effects (KIE) [ Du , X. et al. ( 2004 ) Proc. Natl. Acad. Sci. U.S.A. 101 , 8858 - 8863 ]. To probe the mechanisms of GTPase activation protein (GAP)-facilitated GTP hydrolysis reactions, we measured the (18)O KIEs in GTP hydrolysis catalyzed by Ras in the presence of GAP(334) or NF1(333), the catalytic fragment of p120GAP or NF1. The KIEs in the leaving group oxygens (the beta nonbridge and the beta-gamma bridge oxygens) reveal that chemistry is rate-limiting in GAP(334)-facilitated GTP hydrolysis but only partially rate-limiting in the NF1(333)-facilitated GTP hydrolysis reaction. The KIEs in the gamma nonbridge oxygens and the leaving group oxygens reveal that the GAP(334) or NF1(333)-facilitated GTP hydrolysis reaction proceeds through a loose transition state that is similar in nature to the transition state of the GTP hydrolysis catalyzed by Ras alone. However, the KIEs in the pro-S beta, pro-R beta, and beta-gamma oxygens suggest that charge increase on the beta-gamma bridge oxygen is more prominent in the transition states of GAP(334)- and NF1(333)-facilitated reactions than that catalyzed by the intrinsic GTPase activity of Ras. The charge distribution on the two beta nonbridge oxygens is also very asymmetric. The catalytic roles of active site residues were inferred from the effect of mutations on the reaction rate and KIEs. Our results suggest that the arginine finger of GAP and amide protons in the P-loop of Ras stabilize the negative charge on the beta-gamma bridge oxygen and the pro-S beta nonbridge oxygen of a loose transition state, whereas Lys-16 of Ras and Mg(2+) are only involved in substrate binding.
Du
Xinlin
X
Department of Biochemistry, University of Texas, Southwestern Medical Center, 6001 Forest Park, Room ND10.300, Dallas, Texas 75390-9050, USA.
Sprang
Stephen R
SR
D000818:Animals; D001120:Arginine; D055162:Biocatalysis; D017124:Conserved Sequence; D004583:Electrons; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D010103:Oxygen Isotopes / Q000737:chemistry; D011487:Protein Conformation; D013194:Staining and Labeling; D018631:ras Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D020690:GTPase-Activating Proteins; D010103:Oxygen Isotopes; D006160:Guanosine Triphosphate; D001120:Arginine; D018631:ras Proteins