Transition State Structures and the Roles of Catalytic Residues in GAP-Facilitated GTPase of Ras As Elucidated by 18O Kinetic Isotope Effects

Ras is the prototypical member of the small G protein super-family that mediates diverse signaling pathways in eukaryotes (1, 2). Like other G proteins, Ras alternates between the guanosine 5′ diphosphate (GDP1)-bound inactive state and the GTP-bound active state. GTP-bound Ras is capable of binding to different effector proteins and thereby activating multiple signaling pathways. The intrinsic GTPase activity of Ras is very low, necessitating the down-regulation of Ras activity in vivo by GTPase activating proteins (GAPs) (3), including p120GAP (4) and neurofibromin 1(NF1) (5). Both GAPs increase the rate of GTP hydrolysis by more than 105-fold, from 10−4 s−1 to at least 8 s−1 (6, 7). Mutations in Ras that impair either intrinsic or GAP-facilitated GTPase activity leave Ras in a prolonged state of activation and were found in 30% of cancers (8). Mutations in NF1 are responsible for the pathogenesis of neurofibromatosis type 1 (9). The chemical mechanism of Ras-catalyzed GTP hydrolysis, in particular the nature of the transition state in the intrinsic or GAP-facilitated GTPase reaction, has been the focus of considerable debate. The traditional view holds that the hydrolysis of a phosphate monoester with a good leaving group, of which GTP is one example, proceeds through a metaphosphate-like, loose transition state that features extensive cleavage to the bond between the phosphorus and the leaving oxygen but little or no bond formation between the phosphorus and the nucleophile (10–14). Recent computational studies suggest that metaphosphate is generated as a discrete intermediate in the reaction pathway before it is attacked by the nucleophilic water to generate phosphate (15, 16). An alternative, substrate-assisted, mechanism involves a proton transfer from the attacking water to GTP as a pre-equilibrium step (17–19). The hydroxide thus formed then attacks the γ phosphorus to form a phosphorane intermediate, which decays in the rate-limiting step to generate GDP and phosphate. Resolution of the mechanism is the key to the elucidation of the catalytic roles of the enzyme residues in GTP hydrolysis. Infrared and Raman spectroscopic measurements clearly revealed that, upon binding to Ras, the Pβ–Oβ nonbridge bonds of GTP are weakened, but Pγ–Oγ nonbridge bonds are strengthened (20–22). If the distortion of the electronic structure of GTP by Ras represents destabilization of the ground-state in the direction of the transition state, it can be inferred that the transition state is loose. Indeed, our earlier studies revealed substantial 18O KIE in the β–γ bridge oxygen and β nonbridge oxygens in Ras-catalyzed GTP hydrolysis, demonstrating substantial cleavage of Pγ–Oβγ bridge and Pβ–Oβ nonbridge bonds at the transition state (23). KIE in the two β nonbridge oxygens indicated that charge generated by the cleavage of Pγ–Oβγ bridge bond is delocalized into the two β nonbridge oxygen atoms through resonance structures. The KIE data, together with the crystal structures of Ras in complex with various GTP analogues (24, 25), provide insight into how Ras catalyzes GTP hydrolysis. Charge increase on the leaving group oxygens, namely, the β–γ bridge and the two β nonbridge oxygens, indicates that the interactions of Ras with the three leaving group oxygen atoms become stronger at the transition state and thereby afford preferential stabilization of the transition state. These interactions are mainly mediated by the P loop of Ras: the amide hydrogen of Gly-13 forms a short hydrogen bond with the β–γ bridge oxygen; the main chain NH groups of Val-14,Gly-15, and Lys-16, and the side chain of Lys-16 interact with the pro-S β nonbridge oxygen; the main chain NH of Ser-17 and Mg2+ interacts with the pro-R β oxygen (Figure 1). By contrast, interactions between the γ nonbridge oxygens and the Mg2+, the main chain NH groups of Thr-35 and Gly-60, and the side chain of Lys-16 are not expected to provide electrostatic stabilization of the transition state over the ground state. Figure 1 Active site of Ras in complex with GAP334 and GDP · AlF3, a transition state analogue, in the crystal structure of Ras · GDP · AlF3 3 RasGAP (PDB ID 1WQ1). O1, O2, and O3 refer to the pro-S β nonbridge oxygen, the pro-R ... Structural, biochemical, and computational studies led to the notion that GAP334 facilitates the GTPase of Ras by stabilizing a catalytically competent conformation of Ras and by providing an arginine residue to electrostatically stabilize the transition state (13, 26–28). The crystal structure of Ras in complex with GAP334, a catalytic fragment of p120GAP, and GDP · AlF3, a transition state analogue (Figure 1), revealed that GAP334 stabilizes Switch II of Ras in a catalytically competent conformation (26). In this conformation, the main chain carbonyl oxygen of Thr-35 of Ras and the side chain oxygen atom of Gln-61 of Ras, which points away from the γ phosphoryl group in the absence of GAP334, form hydrogen bonds with the nucleophilic water and orient it for inline attack on the γ phosphorus. In addition, Arg-789 of GAP334, often referred to as the arginine finger, extends into the active site of Ras to interact directly with the β–γ bridge oxygen and one of the γ nonbridge oxygen atoms. The critical role of the arginine finger in catalysis was confirmed by biochemical and computational studies (27, 28). NF1333, the catalytic fragment of NF1, is structurally similar to GAP334 and also possesses a catalytic arginine finger (27, 29). Therefore, NF1 was proposed to facilitate GTP hydrolysis by a similar mechanism (29). Here, we address the mechanism of GAP-facilitated GTP hydrolysis using 18O KIE as a probe of the transition state structures in these reactions. We have synthesized a suite of 18O-labeled GTP molecules (Figure 2) that allow the determination of the KIEs in the oxygen atoms at or geminal to the scissile bond. The KIE in γ18O3-labeling is equivalent to the secondary KIE in the hydrolysis of phosphate monoesters. The charge on the β–γ bridge oxygen due to the cleavage of Pγ–Oβγ bridge bond is delocalized into the two β nonbridge oxygens. The electron delocalization effectively increases the bond order of the β–γ bridge oxygen but decreases the bond order of the two β nonbridge oxygen atoms. Therefore, the KIE in β18O3, instead of the KIE in the β–γ bridge oxygen alone, better reflects the extent of cleavage to the Pγ–Oβγ bridge at the transition state (23). The KIE in the β–γ bridge oxygen is determined by the ratio of the KIE in β18O3 to that in β18O2 or by the ratio of KIE in γ18O4 to that in γ18O3.KIEs in the β–γ bridge, the pro-S β, and the pro-R β oxygens afford stereospecific probes of changes in bond orders and thereby the changes in the charges on these atoms at the transition state. Analysis of the kinetic behavior and KIEs of certain Ras mutants has enabled us to address the roles of the highly conserved residues, Lys-16 andGln-61, in the catalytic site of Ras. In experiments with an arginine finger mutant ofNF1333, we have assessed the role of the arginine finger in GAP-facilitated GTP hydrolysis. Figure 2 Double-labeled nucleotides used in this study. Carbon, nitrogen, oxygen, and phosphorus atoms are colored in silver, blue, yellow, and red, respectively. 18O-labels are indicated by *.