Kinetic isotope effects in Ras-catalyzed GTP hydrolysis: Evidence for a loose transition state

Abstract

A remote labeling method has been developed to determine 18 O kinetic isotope effects (KIEs) in Ras-catalyzed GTP hydrolysis. Substrate mixtures consist of 13 C-depleted GTP and [ 18 O, 13 C]GTP that contains 18 O at phosphoryl positions of mechanistic interest and 13 C at all carbon positions of the guanosine moiety. Isotope ratios of the nonvolatile substrates and products are measured by using a chemical reaction interface͞isotope ratio mass spectrometer. The isotope effects are 1.0012 (0.0026) in the ␥ nonbridge oxygens, 1.0194 (0.0025) in the leaving group oxygens (the ␤-␥ oxygen and the two ␤ nonbridge oxygens), and 1.0105 (0.0016) in the two ␤ nonbridge oxygens. The KIE in the ␤-␥ bridge oxygen was computed to be 1.0116 or 1.0088 by two different methods. The significant KIE in the leaving group reveals that chemistry is largely rate-limiting whereas the KIEs in the ␥ nonbridge oxygens and the leaving group indicate a loose transition state that approaches a metaphosphate. The KIE in the two ␤ nonbridge oxygens is roughly equal to that in the ␤-␥ bridge oxygen. This indicates that, in the transition state, Ras shifts one-half of the negative charge that arises from P ␥-O␤-␥ fission from the ␤-␥ bridge oxygen to the two ␤ nonbridge oxygens. The KIE effects, interpreted in light of structural and spectroscopic data, suggest that Ras promotes a loose transition state by stabilizing negative charge in the ␤-␥ bridge and ␤ nonbridge oxygens of GTP. R as is the prototypical member of the family of small G proteins, which along with G␣ subunits of heterotrimeric G proteins, constitute a class of GTP hydrolases that regulate diverse signaling pathways in eukaryotes (1). Ras orchestrates multiple signaling pathways and regulates cell differentiation, proliferation, and apoptosis (2-4). The GTP-bound forms of G proteins are functionally active: that is, they bind to ''effector'' molecules and regulate their activities or location within the cell. Hydrolysis of GTP results in deactivation and effector release (5). In the absence of other factors, the duration of the active signaling state depends on the intrinsic hydrolytic rate of the G protein, which is typically very slow. However, Ras and other G proteins are subject to specific regulation by GTPase-activating proteins (GAPs), which accelerate intrinsic hydrolytic rates by factors ranging from 10 to 10 5 . In particular, RasGAP increases the GTPase rate of Ras by a factor of 10 5 , from 10 Ϫ4 s Ϫ1 to 10 s Ϫ1 (6). Mutations that impair either intrinsic or GAP-facilitated GTPase activity leave Ras in a prolonged state of activation, which is responsible for its role in oncogenic diseases (7).Ras catalyzes the in-line attack of water on the ␥ phosphate of GTP with inversion of configuration (8). However, the nature of the transition state and the rate-limiting step of Ras-catalyzed GTP hydrolysis remain unclear (9-16). A phosphoryl transfer reaction may either proceed through a metaphosphate or a phosphorane intermediate, or by a concerted pathway (Fig. 1) (17, 18...

Mechanistic evidence for a front-side, SNi-type reaction in a retaining glycosyltransferase

“…Subsequently, the distance between UDP and the anomeric carbon determined from this structure might also be an over-or under-estimation. These suggested that the C-O bond to the leaving group was almost broken and that there was asymmetric hydrogen bonding or partial protonation of the leaving group oxygen (or possibly delocalization/non-bridging oxygen protonation 42 ) at an early stage. Such hydrogen-bonding/protonation of the leaving group was again consistent with the observation of hydrogen bonding in the crystal structure of the ternary complex.…”

“…With a phosphate leaving group, negative charge delocalization to the non-bridging oxygens or protonation instead on these non-bridging oxygens may also contribute to the observed lowering of the 18 O leaving group KIE. 42 It may be that protonation and charge delocalization competed for the electron density liberated by the bond cleavage and the KIE of 1.024 determined here reflected both contributions, protonation and charge delocalization. Partial protonation (which was estimated to be less than 50% from the observation of Bronsted and Taft relationships, see below) appeared also to be consistent with the structure of the ternary complex and the model that hydrogen bonding between leaving group and nucleophile guides the latter to the same face as the former 22 .…”

Role of the arginine finger in Ras·RasGAP revealed by QM/MM calculations

“…The results were interpreted as caused by electron charge shift towards the b-phosphate due to enzyme binding, i.e. a charge distribution close to the product state [8,11]. This seems to be a common feature of proteins of the Ras superfamily, as for example tr-FTIR measurements of the RapAERapGAP complex demonstrate a similar behaviour [12].…”

Allosteric Modulation of Ras-GTP Is Linked to Signal Transduction through RAF Kinase

“…These residues are near the ␥-phosphate and oxygen atom that bridges the ␤-and ␥-phosphate groups in GTP. The dissociative-like reaction mechanism observed in the presence and absence of GAP (22,23) requires accumulation of negative charge at the ␤-␥-bridging oxygen atom of GTP in the transition state of the hydrolysis reaction (24). Stabilization of this charge in the GAP-catalyzed reaction is aided by the arginine finger (Arg 789 ), which GAP inserts in the active site, and the ordering of both switch I and switch II at the Ras/RasGAP interface (9).…”