Ras-catalyzed hydrolysis of GTP: a new perspective from model studies.
Despite the biological and medical importance of signal transduction via Ras proteins and despite considerable kinetic and structural studies of wild-type and mutant Ras proteins, the mechanism of Ras-catalyzed GTP hydrolysis remains controversial. We take a different approach to this problem: the uncatalyzed hydrolysis of GTP is analyzed, and the understanding derived is applied to the Ras-catalyzed reaction. Evaluation of previous mechanistic proposals from this chemical perspective suggests that proton abstraction from the attacking water by a general base and stabilization of charge development on the gamma-phosphoryl oxygen atoms would not be catalytic. Rather, this analysis focuses attention on the GDP leaving group, including the beta-gamma bridge oxygen of GTP, the atom that undergoes the largest change in charge in going from the ground state to the transition state. This leads to a new catalytic proposal in which a hydrogen bond from the backbone amide of Gly-13 to this bridge oxygen is strengthened in the transition state relative to the ground state, within an active site that provides a template complementary to the transition state. Strengthened transition state interactions of the active site lysine, Lys-16, with the beta-nonbridging phosphoryl oxygens and a network of interactions that positions the nucleophilic water molecule and gamma-phosphoryl group with respect to one another may also contribute to catalysis. It is speculated that a significant fraction of the GAP-activated GTPase activity of Ras arises from an additional interaction of the beta-gamma bridge oxygen with an Arg side chain that is provided in trans by GAP. The conclusions for Ras and related G proteins are expected to apply more widely to other enzymes that catalyze phosphoryl (-PO(3)2-) transfer, including kinases and phosphatases.
Maegley
K A
KA
Department of Biochemistry, Beckman Center, Stanford University, CA 94305-5307, USA.
Admiraal
S J
SJ
Herschlag
D
D
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D020558:GTP Phosphohydrolases / Q000737:chemistry*; D019204:GTP-Binding Proteins / Q000737:chemistry*; D020690:GTPase-Activating Proteins; D005998:Glycine / Q000737:chemistry; D006153:Guanosine Diphosphate / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry*; D006860:Hydrogen Bonding; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010710:Phosphates / Q000737:chemistry; D011506:Proteins / Q000502:physiology; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry*; D013329:Structure-Activity Relationship; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D010710:Phosphates; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D016283:Proto-Oncogene Proteins p21(ras); D005998:Glycine